Method for purifying single-stranded RNA

Abstract

A method for purifying single-stranded RNA (ssRNA) is disclosed. More specifically, a method for purifying ssRNA-containing samples containing double-stranded RNA (dsRNA) as an impurity is disclosed using two different chromatographic steps. The method includes the following steps: (a) initial purification of the sample containing both ssRNA and double-stranded RNA (dsRNA) as an impurity using affinity chromatography; and (b) secondary purification of the sample using at least one chromatographic technique selected from the group consisting of size exclusion chromatography (SEC), anion exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC). This method offers high mRNA recovery and dsRNA removal rates of 95% or more, and is particularly useful because it allows for minimizing or appropriately controlling the dsRNA content in mRNA samples, enabling the development of drugs from mRNA regardless of sequence type and maximizing patient safety and drug efficacy.

Description

[Technical Field]

[0001] This invention relates to a method for purifying single-stranded RNA (ssRNA), and more specifically, to a method for purifying a sample containing ssRNA that contains double-stranded RNA (dsRNA) as an impurity using two different chromatographic steps. [Previous Technology]

[0002] It has been reported that during in vitro transcription (IVT) synthesis of mRNA using T7 RNA polymerase (Yin et al., Cell, Vol. 116, pp. 393-404, 2004), a large number of abnormal products, including double-stranded RNA (dsRNA), are produced due to the unconventional activity of the enzyme (Gong et al., JBC, Vol. 281, pp. 23533-23544, 2006). It is known that after entering the human body, dsRNA will initiate antiviral and (TLR3, RIG-I and MDA-5 mediated) inflammatory signaling pathways, leading to a variety of immune 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).

[0003] Representative pattern recognition receptors (PRRs) that recognize dsRNA include TLR3, RIG-I, and MDA-5. TLR3 is located in the endosome and recognizes dsRNAs of 40-50 bp in length; RIG-I is located in the cytosol and recognizes dsRNAs of 500 bp or shorter in length; and MDA-5 is one of the RIG-I-like receptors (RLRs) that recognizes dsRNAs of 2,000 bp or longer in length. When dsRNAs are recognized by TLR3, RIG-I, and MDA-5, type I interferons (IFN, namely IFN-σ and IFN-β) and pro-inflammatory cytokines are secreted. Currently, chemokines, interferon-γ-induced protein 10 (IP-10, CXCL10), and monocyte chemoattractant protein 1 (MCP-1, CCL2) are secreted under the influence of type I IFN and are believed to help recruit 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, YG et al., Nat Rev Mol Cell Biol, Vol. 23, pp. 286-301, 2022).

[0004] Therefore, it is very important to remove dsRNA from IVT mRNA used as a therapeutic agent.

[0005] Several methods are known for removing dsRNA from mRNA formulations. For example, dsRNA can be removed by affinity chromatography on cellulose-based chromatographic media (Baiersdörfer 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). The adsorption mechanism is not fully understood, but dsRNA binds under specific conditions while ssRNA flows through. This method is effective on a laboratory scale, but its drawback is low capacity. Low capacity corresponds to the large column volume required for production scale, which necessitates large buffer volumes, large production areas, and extended process times, thus reducing the productivity of the production equipment. This method also leads to dilution of the treated ssRNA, resulting in a corresponding increase in product volume, which complicates subsequent purification steps.

[0006] dsRNA can also be removed by ion-pair reversed-phase chromatography (RPC) using a styrene-divinylbenzene (SDVB) solid phase (Nwokeoji AO, et al., J. Chromatogr B Analyt Technol Biomed Life Sci. Vol.1104, pp.212-219, 2019). RPC uses toxic and flammable organic solvents, requiring extremely expensive specialized equipment on an industrial scale to mitigate fire and explosion risks. RPC also introduces further safety concerns due to the toxicity of organic solvents in the work environment and the handling of hazardous waste. In addition to solvent issues, RPC separation often requires further temperature increases to obtain optimal results.

[0007] dsRNA can also be removed using size exclusion chromatography (Kim I, et al., RNA, Vol. 13(2), pp. 289-94, 2007). However, the disadvantage of size exclusion chromatography is 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 complex conditions to achieve adequate separation.

[0008] dsRNA can 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 effectiveness in removing DNA and protein contaminants from larger mRNAs (1,000-10,000 bases) and is only effective at elevated operating temperatures. Raising the temperature to 65ºC allows for the elution of larger mRNAs in a sodium chloride gradient. However, high-temperature operation introduces complex logistics challenges, as buffers, samples, and columns must be pre-equilibrated to the specified operating temperature and precisely maintained at that temperature throughout the process, and reproducible across all batches over the entire manufacturing cycle of the product (which can take years).

[0009] Hyperphobic interaction chromatography can also be used to remove dsRNA (Pete Gagnon et al., Cell and Gene therapy insights. Vol.6(7), pp.1035-1046, 2020). However, the disadvantages of hydrophobic interaction chromatography are low purity of the purified product and long purification time.

[0010] dsRNA can also be removed using E. coli RNase III, an enzyme that specifically hydrolyzes dsRNA but not ssRNA (WO 2013 / 102 203 A1). However, RNase III may induce undesirable reactions (such as undesirable immune responses) in patients receiving RNA therapy. Therefore, enzyme removal is required before RNA is administered to patients, which increases the complexity and cost of the method. Furthermore, the use of RNase III often results in partial degradation of ssRNA (especially long ssRNA) during incubation.

[0011] Therefore, the inventors have made great efforts to solve the above problems and developed a purification method with high ssRNA recovery rate and improved dsRNA removal rate. It was found that when initial purification is performed using affinity chromatography, followed by secondary purification using size exclusion chromatography, anion exchange chromatography or hydrophobic interaction chromatography, ssRNA with 95% or more of dsRNA removed can be obtained while maintaining a yield of about 80% or more, thus completing the present invention. [Summary of the Invention]

[0012] The purpose of this invention is to provide a method for purifying single-stranded RNA (ssRNA) by specifically removing dsRNA as an impurity.

[0013] To achieve the above objective, the present invention provides a method for purifying single-stranded RNA (ssRNA), comprising the following steps: (a) performing preliminary purification of a sample containing ssRNA and double-stranded RNA (dsRNA) as impurities using affinity chromatography; and (b) performing secondary purification of the sample using at least one chromatographic method selected from the group consisting of size exclusion chromatography (SEC), anion exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC).

Implementation Method

[0029] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Generally, the terms used in this specification and the experimental methods described below are well-known and commonly used in the art.

[0030] In this invention, it was found that when the product obtained by IVT is initially purified by affinity chromatography and then purified a second time using the differences in size, charge and hydrophobicity between the product and ssRNA (which distinguishes dsRNA containing poly-A tails, i.e. impurities), the removal rate of dsRNA is improved.

[0031] That is, in one embodiment of the present invention, it was demonstrated that when the mRNA product obtained by IVT is initially purified by affinity chromatography based on oligo-dT, and then purified a second time by size exclusion chromatography, anion exchange chromatography or hydrophobic interaction chromatography, the removal rate of dsRNA is improved while the high recovery rate of ssRNA is maintained (Figure 1).

[0032] Therefore, in one aspect, the present invention relates to a method for purifying single-stranded RNA (ssRNA), comprising the following steps:

[0033] (a) Preliminary purification of samples containing ssRNA and double-stranded RNA (dsRNA) as impurities using affinity chromatography; and

[0034] (b) The sample is further purified using at least one of the group consisting of size exclusion chromatography (SEC), anion exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC).

[0035] In the present invention, the RNA may be, but is not limited to, any type of RNA, and is preferably, but is not limited to, mRNA.

[0036] In the present invention, samples can be obtained by, but are not limited to, in vitro transcription (IVT).

[0037] In the present invention, the dsRNA may be any double-stranded RNA as an IVT result, preferably, but not limited to, a dsRNA containing a poly(A) tail.

[0038] In the present invention, the step (a) of preliminary purification of the sample using affinity chromatography may include the following steps:

[0039] (ai) the sample containing the ssRNA was mixed with the sample preparation buffer and the mixture was loaded into a column containing a resin complementaryly bound to the poly(A) tail;

[0040] (a-ii) washing the column using washing buffer;

[0041] (a-iii) Recovered bound ssRNA by elution with elution buffer.

[0042] In the present invention, the resin complementary to the poly(A) tail may be, but is not limited to, a polythymidine resin (oligodT resin).

[0043] In the present invention, the sample preparation buffer may be a solution containing the following (pH 6.0 to 7.0): 10 to 150 mM, preferably 10 to 100 mM sodium phosphate, tris(hydroxymethyl)aminemethane (tris), sodium citrate or sodium acetate; mM, preferably 100 to 900 mM, more preferably 100 to 800 mM sodium chloride. More preferably, the sample preparation buffer may be a solution containing the following (pH 6.3 to 6.8): 20 to 100 mM sodium phosphate; Most preferably, the sample preparation buffer may be a solution containing the following (pH 6.5): 100 mM sodium phosphate; 10 mM EDTA; and 700 mM sodium chloride, or a solution (pH 6.6) containing the following: 20 mM sodium phosphate; EDTA;and 700 mM sodium chloride.

[0044] In this invention, the term "sample preparation buffer" refers to a buffer solution that adjusts the ionic strength, etc., of a sample so that the sample can be loaded onto a column containing chromatographic resin, and this term is used in the same sense as an n-fold concentrated equilibration buffer. For example, a 2X equilibration buffer is a buffer solution prepared by adding twice the amount of reagents as an equilibration buffer, and can have the same meaning as a sample preparation buffer solution. In the case of a buffer solution containing salt, it will be apparent to those skilled in the art that the pH can vary with the salt concentration.

[0045] In this invention, step (a) may further include an additional loading equilibration buffer step before step (a-ii).

[0046] In this invention, the equilibration buffer may be a solution containing the following (pH 6.0 to 7.0): 10 to 100 mM sodium phosphate, tris(hydroxymethyl)aminomethane, sodium citrate or sodium acetate; 1 to 10 mM EDTA (ethylenediaminetetraacetic acid); and 100 to 500 mM sodium chloride. More preferably, the equilibration buffer may be a solution containing the following (pH 6.5 to 7.0): 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 containing (pH 6.7): 50 mM sodium phosphate; 5 mM EDTA; and 350 mM sodium chloride, or a solution containing (pH 6.8): 10 mM sodium phosphate; 5 mM EDTA; and 250 mM sodium chloride, or a solution containing (pH 6.8): 10 mM sodium phosphate; 5 mM EDTA; and 350 mM sodium chloride. In this invention, the washing buffer may be a solution containing (pH 6.0 to 7.0): 10 to 100 mM sodium phosphate, Tris, sodium citrate, or sodium acetate; and 1 to 10 mM EDTA.

[0047] More preferably, the washing buffer may be a solution containing 50 mM sodium phosphate and 5 mM EDTA (pH 6.7).

[0048] In this invention, the elution buffer may be a solution containing 0.01 to 10 mM sodium phosphate, Tris, sodium citrate, or sodium acetate (pH 6.0 to 7.0). More preferably, the elution buffer may be a solution containing 5 mM Tris (pH 7.0) or a solution containing 5 mM sodium citrate (pH 6.0 to 6.5).

[0049] In this invention, it is obvious that if the pH of the buffer and the concentration of the components are lower or higher than the above values, the chromatographic performance will be reduced and therefore the purified product cannot be obtained with a high recovery rate.

[0050] In this invention, step (b) of secondary purification of the sample using size exclusion chromatography may include the following steps:

[0051] (b-1-i) Mix the pre-purified sample eluted in step (a) with 2X equilibration buffer and load the mixture into a column containing size exclusion resin;

[0052] (b-1-ii) Elute the bound RNA using the equilibration buffer described above; and

[0053] (b-1-iii) Obtain the final eluted RNA as the fraction containing ssRNA.

[0054] In this invention, size exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method for separating molecules based on size and molecular weight. In this invention, size exclusion chromatography can be performed using various size exclusion chromatography resins or columns containing such resins known in the art. For example, size exclusion chromatography resins can typically comprise polymers having fine-pore beads through which substances are separated. In this invention, the polymer can be, for example, dextran, agarose, or polyacrylamide, preferably Superdex™ 200, Superose™ 6, Sepharose™ 6, or Sepharcyl™ S-300, most preferably Superose™ 6, but is not limited thereto.

[0055] In this invention, the size exclusion resin can separate molecules of 10 to 7,000 kDa, more preferably 1,000 to 6,000 kDa, and most preferably 1,500 to 5,000 kDa, depending on their size.

[0056] In this invention, the equilibration buffer may be, but is not limited to, a solution containing (pH 3.0 to 7.0) the following: 1 to 100 mM sodium citrate, sodium phosphate, Tris or sodium acetate; and 0.01 to 1,000 mM sodium chloride, preferably a solution containing (pH 4.0 to 6.5) the following: 10 to 80 mM sodium citrate, sodium phosphate, Tris or sodium acetate; and 0.1 to 700 mM sodium chloride, more preferably a solution containing (pH 4.5 to 5.5) the following: 30 to 70 mM sodium citrate, sodium phosphate, Tris or sodium acetate; and 100 to 500 mM sodium chloride, and most preferably a solution containing 50 mM sodium citrate and 200 mM sodium chloride (pH 5.0).

[0057] In this invention, it is obvious that if the pH of the buffer and the concentration of the components are lower or higher than the above values, the chromatographic performance will be reduced and therefore the purified product cannot be obtained with a high recovery rate.

[0058] In this invention, step (b) of secondary purification of the sample using anion exchange chromatography may include the following steps:

[0059] (b-2-i) The preliminarily purified sample eluted in step (a) is mixed with sample preparation buffer, and the mixture is loaded into a column containing anion exchange resin; and

[0060] (b-2-ii) Collect the eluted flow-through (FT) fractions into fractions containing ssRNA.

[0061] In this invention, ssRNA can be collected using a flow-through mode, wherein the flow-through (FT) eluted by chromatography is collected as a fraction containing ssRNA.

[0062] The terms “flow-through fluid (FT),” “flow-through mode” and “flow-through purification” are used interchangeably herein and refer to a separation technique in which at least one target molecule (e.g., ssRNA) contained in a biopharmaceutical formulation passes through a substance that is normally bound to one or more impurities, while the target molecule is normally not bound (i.e., flow-through).

[0063] In this invention, the term "anion exchange chromatography" refers to a process for separating substances based on charge through an ion exchange resin containing positively charged groups such as diethylaminoethyl (DEAE).

[0064] In the present invention, various commercially available anion exchange chromatography resins can be used. Examples of anion exchange chromatographic resins include, but are not limited to, those anion exchange chromatographic resins substituted with diethylamine ethyl (DEAE), trimethylamine ethyl (TAME), triethylamine ethyl (TEAE), amine ethyl (AE), diethylaminepropyl (ANX), or quaternary ammonium (Q) groups. Preferably, the anion exchange chromatography resin is any one selected from an anion exchange resin having a strong alkaline quaternary ammonium group or an anion exchange resin having a weakly basic diethylamine-ethyl (DEAE) group. More preferably, the anion exchange chromatography resin may be any one selected from the group consisting of an anion exchange resin having a strong alkaline quaternary ammonium (Q) group. Most preferably, the anion exchange chromatography resin may be, but is not limited to, Q Sepharose.

[0065] In the present invention, the sample preparation buffer may be, but is not limited to, a solution containing the following (pH 7.0 to 8.0): 10 to 50 mM Tris, sodium phosphate, sodium citrate or sodium acetate; EDTA;and 700 mM sodium chloride.

[0066] In the present invention, it is obvious that if the pH and fraction concentration of the buffer is below or above the above values, the chromatographic performance is reduced and the purified product cannot be obtained with high recovery.

[0067] In the present invention, the step (b) of secondary purification of the sample using hydrophobic action chromatography may include the following steps:

[0068] (b-3-i) The preliminary purified sample eluted in step (a) is mixed with sample preparation buffer, and said mixture is loaded into a column containing a hydrophobic acting resin;

[0069] (b-3-ii) The column was washed using equilibrium buffer;

[0070] (b-3-iii) Recovered bound ssRNA by elution with elution buffer.

[0071] In the present invention, hydrophobic action chromatography is a method for separating the target molecule based on its degree of hydrophobicity. For example, hydrophobic groups such as phenyl, octyl, and butyl can be attached to the HIC resin (stationary phase) by hydrophobic interaction with the molecule of interest. In the present invention, various commercially available hydrophobic acting chromatographic resins can be used. For example, a hydrophobically acting chromatographic resin may include a hydrophobic moiety (moiety) selected from an alkyl group, an aromatic group, and an ether. More specifically, alkyl groups include lower alkyl groups such as n-propyl, isopropyl, n-butyl, isobutyl and n-octyl, and aromatic groups include, but are not limited to, substituted or unsubstituted phenyl groups. Additionally, the hydrophobically acting chromatographic resin may include a matrix selected from agarose, Sepharose (GE Healthcare), polystyrene, divinylbenzene, and combinations thereof. More preferably, the hydrophobically acting chromatographic resin may be C4-HLD, Benzyl Ultra, or Capto Phenyl, most preferably, but not limited to, C4-HLD resin.

[0072] In the present invention, the sample preparation buffer or equilibrium 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; mM sodium chloride, sodium sulfate, ammonium sulfate, potassium sulfate, disodium phosphate, lithium chloride or potassium thiocyanate, and more preferably solutions (pH 7.0) containing: 50 mM sodium phosphate; 10 mM EDTA;

[0073] In the present invention, step (b) may further include a step of additional loading equilibrium buffer before step (b-3-ii).

[0074] In the present invention, the elution buffer may be, but is not limited to, a solution containing the following (pH 6.5 to 7.5): 10 to 100 mM sodium phosphate, tris, sodium citrate or sodium acetate; EDTA;and 500 mM to 600 mM sodium chloride.

[0075] In the present invention, it is apparent that if the pH and fraction concentrations of the buffer are below or above the above values, the chromatographic performance is reduced and the purified product cannot be obtained with high recovery.

[0076] In this invention, the purification method may further include an ultrafiltration / difiltration (UF / DF) step before, after, or after step (b).

[0077] In this invention, "UF filtration" refers to a technique that uses a permeable filter capable of separating components based on their molecular weight (molecular size) to remove or collect any component (e.g., particles) from a target substance (solution), thereby improving the purity of the target substance. Ultrafiltration / permeation (UF / DF) can be performed using a conventional UF / DF system and may include changes in constant osmotic pressure, buffer replacement, and concentration adjustment.

[0078] In this invention, the ultrafiltration step can be performed by a tangential flow filtration (TFF) method. "Tangential flow filtration (TFF)," also known as "cross-flow filtration," refers to a filtration method in which water and a sample pass tangentially through a membrane.

[0079] In this invention, the ssRNA obtained through secondary purification can have a dsRNA removal rate of 80% or more, more preferably 90% or more, and most preferably 95% or more.

[0080] Example

[0081] The present invention will be described in more detail below through embodiments. These embodiments are merely illustrative of the invention, and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these embodiments.

[0082] Experimental Method 1. Determination of mRNA Concentration

[0083] In the following examples, the mRNA concentration was determined by measuring the absorbance at 260 / 280 nm using UV spectrophotometry (NanoDrop, Thermo Fisher Scientific, USA).

[0084] Experimental Method 2. Measurement of dsRNA Concentration

[0085] In the following examples, the dsRNA concentration was measured in the following manner.

[0086] First, the capture antibody (SCICONS, Netherlands) was diluted to 3 μg / mL in 1x PBS. 100 μL of the dilution was loaded into each well of a 96-well microplate and incubated overnight at 4ºC. The plate was then inverted to discard the solution and remove any residual solution. Subsequently, the process of adding 300 μL of wash buffer (0.5% Tween in PBS) to each well and then discarding the solution was repeated three times, followed by tapping the plate on several layers of paper towels to completely remove any residual solution. After adding 200 μL of blocking buffer (ThermoFisher, USA) to each well, the plate was covered with plate sealer and incubated at 37ºC for 2 hours. Next, the plate was inverted to discard the solution and then remove any residual solution. The process of adding 300 µL of wash buffer to each well and then discarding the solution was repeated three times, followed by tapping the plate on several layers of paper towels to completely remove any residual solution. Add 100 µL of each analyte concentration of standard solution (Abnova, Taiwan) diluted in STE buffer (100 mM NaCl, 50 mM Tris, 1 mM EDTA, pH 7.0) to two copies of the test solution to the wells and incubate at 37ºC for 1 hour. After incubation, invert the plate to discard the solution and then remove any residual solution. Repeat the process of adding 300 µL of wash buffer to each well and discarding the solution three times, then gently tap the plate on several layers of paper towels to completely remove any residual solution.

[0087] The detection antibody (SCICONS, Netherlands) was diluted 1:1 with 1xPBS, and 100 µL of the dilution was loaded in duplicate into each well. The plate was covered with a sealant and incubated at 37ºC for 1 hour. The plate was then inverted to discard the solution, and any residual solution was removed. The process of adding 300 µL of wash buffer to each well and then discarding the solution was repeated three times, and the plate was then tapped on several layers of paper towels to completely remove any residual solution. The HRP-conjugated anti-IgM antibody (EMD Millipore, USA) was diluted 1 / 10,000 in blocking buffer, and 100 µL of the dilution was loaded into each well. The plate was covered with a sealant and incubated at 37ºC for 1 hour. The plate was then inverted to discard the solution, and any residual solution was removed. The process of adding 300 µL of wash buffer to each well and then discarding the solution was repeated three times, and the plate was then tapped on several layers of paper towels to completely remove any residual solution.

[0088] Add 100 µL of a one-step ultra-TMB-ELISA (ThermoFisher, USA) to each well, cover the plate with plate sealant, and incubate at room temperature for 15 minutes. Then, add 100 µL of stop buffer (1 M H2SO4) to each well to terminate the reaction and measure the absorbance at 450 nm.

[0089] After subtracting the blank absorbance from the absorbance of the standard solution and the test solution, a regression equation was obtained using a 4-parameter logistic model with the standard solution concentration as the X-axis and the response value as the Y-axis, thus establishing a standard curve. The dsRNA concentration was calculated by substituting the response value of the test solution into the equation.

[0090] Example 1. Preparation of nucleic acid for synthesizing mRNA containing dsRNA as an impurity.

[0091] 1-1. Linearization of template DNA preparation

[0092] Linearization was achieved by cutting the portion downstream of the polyA site in the plasmid using a restriction endonuclease. The linearized DNA was isolated using AMICON, and plasmid DNA cleavage was then assessed on a 1% agarose gel.

[0093] 1-2. In vitro transcription

[0094] In vitro transcription is the process of synthesizing mRNA.

[0095] The prepared template DNA was reacted with T7 RNA polymerase, buffer, NTPs (including natural and chemically modified NTPs) and other necessary elements for IVT at 37ºC for 4 hours, as shown in Table 1 below.

[0096] [Table 1] reagents IVT volume (μL) supplier Catalog Number water 253 - - ATP 800 Roche 04980824103 CTP 800 Roche 04980875103 GTP 800 Roche 04980859103 m1ΨTP 800 Thermo R0491SKB012 Hat-like objects 640 TriLink N-7413 reaction buffer 800 self made - Template DNA 1262 self made - RI 200 Roche 09537589103 PPase 20 Roche 08140677103 T7 RNAP 1625 Thermo EP011SKB011

[0097] After the reaction is complete, 1 μg of DNA is treated with 1 U of DNase I and then reacted at 37ºC for 15 to 30 minutes to remove the template DNA for subsequent purification processes.

[0098] Example 2. Preliminary purification using affinity chromatography

[0099] The IVT product obtained in Example 1 was initially purified using a column containing oligo(A) tail bound to oligo(dT).

[0100] The specific methods are summarized in Table 2 below.

[0101] [Table 2] parameter affinity resin Integral column oligomer dT Dynamically combined capacity Approximately 3 mg / mL of resin Flow rate ≤1.5 CV / min (Sample loading: ≤1 CV / min) Rebalancing steps Volume: ≤ 10 CV Washing steps Volume: ≤ 40 CV Washing steps Based on 20 mAu Clean in place (CIP) 30 min ≤ Buffer composition EQ: 50 mM sodium phosphate + 350 mM sodium chloride + 5 mM EDTA, pH 6.7 Washing: 50 mM sodium phosphate + 5 mM EDTA, pH 6.7 Elution: 5 mM Tris, pH 7.0 CIP: 0.5 N sodium hydroxide

[0102] Specifically, an 8 mL (2 µm) (Sartorius, USA) column of CIMmultus oligo-dT (C12 adapter) was mounted onto an AKTA Avant 150 (Cytiva, USA), and 2.5 mg / mL of the IVT product from Example 1, mixed with 3 mL of sample preparation buffer (100 mM sodium phosphate + 700 mM NaCl + 10 mM EDTA, pH 6.5), was loaded into the column at the flow rate shown in Table 2 above. The column was then reequilibrated with 5 CV of equilibration buffer, and the column containing the bound product was washed with 40 CV or less of wash buffer. Then, 5 CV of elution buffer was loaded to recover the IVT product containing the poly(A) tail bound to the column. Afterward, the column was washed with CIP buffer, and the concentrations of mRNA and dsRNA were measured according to the descriptions in Experimental Methods 1 and 2 above.

[0103] Example 3. Secondary purification and effect evaluation using size exclusion chromatography (SEC)

[0104] The IVT described in Example 1 can generate many types of dsRNA, but they can be roughly divided into three categories. First, if the invalid transcript generated during mRNA synthesis has a complementary sequence, it can bind to mRNA to generate dsRNA. In this case, the formed dsRNA has a poly(A) tail.

[0105] Furthermore, if a T7 promoter-like sequence is present in the non-template DNA, a reverse transcript may be generated. If a poly(A) tail is present, the likelihood of generating dsRNA through antisense transcription recognizing the non-template DNA is lower, but since a promoter-independent reverse transcript can be generated, it is possible to generate dsRNA.

[0106] Furthermore, IVT terminates via run-off transcription, which can result in mRNAs that are longer than the original mRNA. The longer mRNAs are circular dsRNAs generated by forming a hairpin structure at the 3' end and extending through their own primers, and have poly(A) tails.

[0107] Since most of the aforementioned dsRNAs have poly(A) tails, they cannot be removed using the oligodT process. Furthermore, since dsRNAs are formed by attaching complementary sequences to mRNA, they are expected to be larger than mRNAs. Considering this, the inventors attempted to use size exclusion resins to detect dsRNA removal rates.

[0108] The properties of the size exclusion resins used in the following embodiments are shown in Table 3 below.

[0109] [Table 3]

[0110] 3-1. Secondary purification and effect evaluation using Superdex 200 PG resin

[0111] The secondary purification process using Superdex 200 PG resin is summarized in Table 4 below.

[0112] [Table 4] parameter Superdex 200 PG Flow rate 2.6 mL / min (approximately 30 cm / hr) Sample concentration 1,011 µg / mL Sample injection volume 7 mL Total Samples 7.2 mg Column volume 319 mL The ratio of sample loading to column volume 2.2% EQ / elution buffer 1 mM citrate + 200 mM NaCl, pH 6.0 to 6.4

[0113] That is, the preliminary purified product obtained in Example 2 was subjected to UFDF (3,000 g for 20 min) using Amicon (Merck, Germany), and then mixed with the 2X equilibration buffer shown in Table 4 to prepare the injection sample. A Hiload 26 / 600 Superdex 200 pg (Cytiva, USA) column was mounted onto an AKTA Avant 150 (Cytiva, USA), and a sample 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 passed through, and the eluted flow-through (FT) fraction was collected. The total mRNA and dsRNA concentrations in each fraction were measured according to Experimental Methods 1 and 2.

[0114] The results are shown in Table 5 and Figure 2 below, confirming that although the peaks on the chromatogram were not separated, dsRNA was distributed in the prefraction as expected, and the dsRNA removal rate was about 72% when the fraction was collected based on a 79% mRNA recovery rate.

[0115] In the following embodiments, the mRNA recovery rate and dsRNA removal rate were calculated as follows.

[0116] Collected fractions = Fractions obtained after removing the pre-fractions containing a large amount of dsRNA.

[0117] mRNA recovery rate (%) = (mRNA content of collected fraction / mRNA content of loaded fraction) * 100

[0118] dsRNA removal rate (%) = (1 - (dsRNA content of collected fraction / dsRNA content of loaded fraction)) * 100

[0119] For example, the recovery rate in Table 5 below is calculated as follows.

[0120] mRNA recovery rate (%) = (Fr. 11 - Fr. 45 mRNA content / loaded mRNA content) * 100

[0121] dsRNA removal rate (%) = (1 - (Fr. 11 - Fr. 45 mRNA content / loaded mRNA content)) * 100

[0122] [Table 5]

[0123] 3-2. Secondary purification and effect evaluation using Superose 6 PG resin

[0124] As demonstrated in Example 3-1, dsRNA is larger than ssRNA, and the SEC column exhibits a higher dsRNA removal rate than other columns. However, to further improve the dsRNA removal rate, a secondary purification was performed using Superose 6 PG resin with a wider operating range, and the pH and salt concentration were adjusted to further enhance the dsRNA removal rate. This method is summarized in Table 6 below.

[0125] [Table 6] parameter Superose 6 PG Flow rate 1 mL / min (approximately 30 cm / hr) Sample concentration 760 µg / mL Sample injection volume 2 mL Total Samples 1.5mg Column volume 121 mL The ratio of sample loading to column volume 1.7% EQ / elution buffer Run a solution of 1:50 mM citrate + 200 mM NaCl, pH 5.0. Run 2: 50 mM citrate + 1000 mM NaCl, pH 5.0 Run 3: 50 mM Tris + 200 mM NaCl, pH 7.5 Run 4: 50 mM Tris + 1000 mM NaCl, pH 7.5

[0126] That is, the preliminary purified product obtained in Example 2 was subjected to UFDF (3000 g for 20 min) using Amicon (Merck, Germany), and then mixed with the 2X equilibration buffer shown in Table 6 to prepare the injection sample. A HiLoad 16 / 600 Superose 6 pg (Cytiva, USA) column was loaded into an AKTA Avant 150 (Cytiva, USA), and the sample at a concentration of 760 μg / mL was injected into the column at a rate of 1 mL / min under the conditions shown in Table 6. Then, 2 CV (column volume) of equilibration buffer was passed through, and the eluted flow-through (FT) fraction was collected. The total mRNA and dsRNA concentrations in each fraction were measured according to Experimental Methods 1 and 2.

[0127] The results are shown in Table 7 and Figure 3 below, confirming that the dsRNA peak is concentrated at the front, similar to the case with Superdex 200 pg. The higher the pH and the higher the salt concentration, the larger the front peak size. Furthermore, it is further confirmed that as the front peak on the chromatogram becomes smaller, the mRNA recovery and dsRNA removal rates can be maximized when collecting the later peaks (Run 1 in Table 7).

[0128] That is, as shown in Table 7 below, the results of run 1 with the smallest front peak indicate that the dsRNA removal rate is 97% and the mRNA recovery rate is 79%.

[0129] [Table 7]

[0130] 3-3. Secondary purification and effect evaluation using Sepharose 6 FF resin and Sephacryl S-300 HR resin

[0131] Secondary purification was performed using Sepharose 6 FF, which has a similar operating range to Superose 6 PG used in Examples 3-2, but with larger resin bead sizes; and secondary purification was performed using Sephacryl S-300 HR, which has a narrower operating range, but with similar bead sizes. The methods are summarized in Table 8 below.

[0132] [Table 8] parameter Sepharose 6 FF Sephacryl S-300 HR Flow rate 1 mL / min (approximately 30 cm / hr) 1 mL / min (approximately 30 cm / hr) Sample concentration 725 µg / mL 725 µg / mL Sample injection volume 2 mL 2 mL Total Samples 1.5 mg 1.5 mg Column volume 121 mL 121 mL The ratio of sample loading to column volume 1.7% 1.7% EQ / elution buffer Run a solution of 1:50 mM citrate + 200 mM NaCl, pH 5.0. Run a solution of 1:50 mM citrate + 200 mM NaCl, pH 5.0. Run 2: 50 mM Tris + 1000 mM NaCl, pH 7.5 -

[0133] That is, the preliminary purified product obtained in Example 2 was subjected to UFDF (3000 g for 20 minutes) using Amicon (Merck, Germany), and then mixed with the 2X equilibration buffer shown in Table 6 to prepare the injection sample.

[0134] With Sepharose 6 FF, a Sepharose 6 Fast Flow (Cytiva, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and a sample 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 passed through, and the eluted flow-through (FT) fraction was collected. The total mRNA and dsRNA concentrations in each fraction were measured according to Experimental Methods 1 and 2. With Sephacryl S-300 HR, a Sephacryl S-300 HR (Cytiva, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and a sample 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 passed through, and the eluted flow-through (FT) fractions were collected. The total mRNA and dsRNA concentrations in each fraction were measured according to Experimental Methods 1 and 2.

[0135] The results, as shown in Figure 4, confirm that, similar to Example 3-2, with Sepharose 6 FF resin, the peak increases with increasing pH and salt concentration. As shown in Figure 5, it is confirmed that with Sephacryl S-300 HR resin, even under the conditions of highest dsRNA removal rate in Example 3-2, the peak was not separated.

[0136] 3-4. Secondary purification and effect evaluation using Superose 6 PG resin and Sepharose 6 FF resin under the same conditions.

[0137] The results of repeating the same process under the conditions of Run 1 in Example 3-2 and Run 1 in Example 3-3 with Sepharose 6 FF resin, as shown in Figure 6, confirmed that the Sepharose 6 FF resin had a higher initial peak, indicating that Superose 6 PG resin was the most effective in ensuring mRNA recovery and dsRNA removal.

[0138] The results of Examples 3-1 to 3-3 are summarized in Table 9 below.

[0139] [Table 9] resin granularity (µm) Scope of work (Spherical, kDa) Scope of work (Dextran, kDa) Are the peaks separated? Superdex 200 pg 24 to 44 10 to 600 1 to 100 X Superose 6 pg 30 to 40 5 to 5,000 N / A O (Separation) Sepharose 6 FF Below 90 10 to 4,000 10 to 2,000 O (Slight separation) Sephacryl S-300 HR Below 50 10 to 1,500 2 to 400 X

[0140] That is, since the dsRNA removal rate is inversely proportional to the mRNA yield, it has been confirmed that under the conditions of secondary purification in the SEC process using Superose 6 PG resin and equilibration and elution buffer composition of 50 mM citrate + 200 mM NaCl, pH 5.0, the dsRNA removal rate is guaranteed to be 95% or higher, while maintaining the mRNA recovery rate at approximately 80% or higher.

[0141] Example 4. Secondary purification and effect evaluation using anion exchange chromatography (AEX).

[0142] It is known that the AEX process removes dsRNA from IVT products, but this process involves binding ssRNA to a column and removing it through washing. However, based on the principle of AEX, it is expected that the efficiency of this process can be improved by binding dsRNA to the column and collecting ssRNA through FT, while controlling the type and concentration of salt. Therefore, the AEX process was performed in FT mode, and the dsRNA removal rate was measured.

[0143] Compared to using the elution mode, the advantage of using the FT mode is that it allows for a significantly increased capacity. For example, assuming the loaded sample contains 0.5% dsRNA, approximately 200 times the capacity can be used. Furthermore, since the elution step can be omitted, it saves on process time and reagents.

[0144] The specific AEX conditions are shown in Table 10 below.

[0145] [Table 10] parameter AEX resin (strong) Q Sepharose FF Resin type beads Flow rate Different each time it runs Column volume 4.7 mL Elution type Different each time it runs Process steps (buffer solution mixing ratio): Volume Buffer composition

[0146] That is, the preliminary purified product obtained in Example 2 is mixed with the 2X equilibration buffer shown in Tables 11 to 18 below to prepare the injection sample.

[0147] A HiScreen Q FF (Cytiva, USA) column was mounted on an 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 experimental methods 1 and 2.

[0148] [Table 11] Run 1 Sample loading amount 2.2mg Flow rate 5 mL / min, elution 5 mL / min Washing methods gradient EQ buffer Buffer A 90%, Buffer B 10% → 0.2 M ammonium sulfate Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 0 mM ammonium sulfate Buffer B: Tris 20 mM + EDTA 5 mM + 2.0 M ammonium sulfate

[0149] First, the results of AEX under the conditions shown in Table 11 confirmed that 161 µg of mRNA was obtained in the flow-through buffer, no mRNA was detected in the elution, and a peak of 2,000 mAU or higher appeared in the CIP. This was thought to be because the mRNA was strongly bound to the resin and therefore did not appear in the FT. Therefore, AEX was performed under the conditions shown in Table 12 below, where the binding force between the mRNA and the resin was relatively weakened by increasing the ionic strength of the EQ buffer.

[0150] [Table 12] Run 2 Sample loading amount 1.2 mg Flow rate 5 mL / min, elution 1 mL / min Washing methods gradient EQ buffer Buffer A 50%, Buffer B 50% -> 1M Ammonium Sulfate Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 0 mM ammonium sulfate Buffer B: Tris 20 mM + EDTA 5 mM + 2.0 M ammonium sulfate

[0151] The results confirmed that mRNA was still not detectable in the flow-through and elution buffers, and a peak of 3,000 mAU or higher appeared in the CIP. In other words, it was confirmed that the mRNA was still strongly bound to the resin and therefore did not appear in the FT. Therefore, AEX was performed using sodium chloride with an ionic strength lower than that of ammonium sulfate under the conditions shown in Table 13 below.

[0152] [Table 13] Run 3 Sample loading amount 1.1 mg Flow rate 5 mL / min, elution 1 mL / min Washing methods gradient EQ buffer Buffer A 87.5%, Buffer B 12.5% ​​→ 200 mM Sodium Chloride Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 0 M sodium chloride Buffer B: Tris 20 mM + EDTA 5 mM + 2.5 M sodium chloride

[0153] The results confirmed that 35 µg of mRNA was measured in the elution buffer, with the remainder corresponding to the peaks observed in CIP. That is, in 200 mM sodium chloride, the mRNA was strongly bound to the resin and therefore did not appear in FT. Therefore, the ionic strength of the EQ buffer was increased, and AEX was performed under the conditions shown in Table 14 below.

[0154] [Table 14] Run 4 Sample loading amount 1.0 mg Flow rate 5 mL / min, elution 1 mL / min Washing methods gradient EQ buffer Buffer A 75%, Buffer B 25% -> 500 mM sodium chloride Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 0 M sodium chloride Buffer B: Tris 20 mM + EDTA 5 mM + 2.5 M sodium chloride

[0155] The results confirmed that 123 µg was measured in the eluent and 322 µg was measured in the eluent (pH 10), with the remainder corresponding to the peaks in the CIP. The combined recovery in the eluent was 44.5% (445 µg), indicating that the mRNA was strongly bound to the resin and therefore the recovery was insufficient. Therefore, AEX was performed under the conditions shown in Table 15 below, where the ionic strength was further increased.

[0156] [Table 15] Run 5 Sample loading amount 0.95 mg Flow rate 5 mL / min, elution 1 mL / min Washing methods gradient EQ buffer Buffer A 76%, Buffer B 24% → 600 mM Sodium Chloride Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 0 M sodium chloride Buffer B: Tris 20 mM + EDTA 5 mM + 2.5 M sodium chloride

[0157] The results confirmed that 661 µg was measured in the eluent, with the remainder corresponding to the peaks observed in CIP. The combined recovery rate in the eluent was 70% (661 µg), indicating that the mRNA was strongly bound to the resin and therefore could not be fully recovered. Therefore, AEX was performed under the conditions shown in Table 16 below, where the ionic strength was further increased.

[0158] [Table 16] Run 6 Sample loading amount 0.9 mg Flow rate 5 mL / min, elution 1 mL / min Washing methods gradient EQ buffer Buffer A 72%, Buffer B 28% -> 700 mM Sodium Chloride Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 0 M sodium chloride Buffer B: Tris 20 mM + EDTA 5 mM + 2.5 M sodium chloride

[0159] The results confirmed that 719 µg was measured in the flow-through solution, with the remainder corresponding to the peaks observed in CIP. As shown in Table 17 below, the mRNA recovery rate was confirmed to be 80%, which is at an appropriate level; the dsRNA removal rate was also 55%, which is the same as or higher than when using the elution mode.

[0160] [Table 17]

[0161] To examine the mRNA recovery rate and dsRNA removal rate under higher salt conditions, AEX was performed under the conditions shown in Table 18 below.

[0162] [Table 18] Running 7 Sample loading amount 0.9 mg Flow rate 5 mL / min, elution 1 mL / min Washing methods gradient EQ buffer Buffer A 68%, Buffer B 32% -> 800 mM sodium chloride Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 0 M sodium chloride Buffer B: Tris 20 mM + EDTA 5 mM + 2.5 M sodium chloride

[0163] The results confirmed that 672 µg was measured in the flow-through solution, with the remainder corresponding to the peaks observed in the CIP. This corresponds to a 75% recovery, which is not significantly different from the results obtained using 700 mM sodium chloride. Therefore, to confirm the results at higher ionic strengths, AEX was performed under the conditions shown in Table 19 below.

[0164] [Table 19] Run 8 Sample loading amount 1.2 mg Flow rate 5 mL / min, elution 1 mL / min Washing methods gradient EQ buffer Buffer A Buffer composition Buffer A: Tris 20 mM + EDTA 5 mM + 1 M sodium chloride Buffer B: Tris 20 mM + EDTA 5 mM + 2.5 M sodium chloride

[0165] The results confirmed that 1 mg of mRNA was measured in the flow-through solution. This is equivalent to an 85% recovery rate, which is not significantly different from the results obtained using 700 mM or 800 mM sodium chloride.

[0166] Example 5. Secondary purification and effect evaluation using hydrophobic interaction chromatography

[0167] Since the structure of mRNA is different from that of dsRNA, it is expected that their hydrophobicity will also be different. Therefore, the inventors attempted to demonstrate the possibility of using HIC resin to remove dsRNA.

[0168] The properties of the hydrophobic resins used in the following examples are shown in Table 20 below.

[0169] [Table 20]

[0170] 5-1. Secondary purification using C4-HLD resin and its effect evaluation

[0171] The secondary purification process using C4-HLD resin is summarized in Table 21 below.

[0172] [Table 21] parameter HIC resin (weak to moderate) C4-HLD Resin type integral column Flow rate 1 CV / min (4 mL / min) Column volume 4 mL Elution type gradient Step by step Process steps (buffer solution mixing ratio): Volume EQ (A: 80%, B: 20%): 10 CV EQ (A: 80%, B: 20%): 10 CV Re-EQ (A: 80%, B: 20%): until UV baseline. Re-EQ (A: 80%, B: 20%): until UV baseline. Elution (A: 80%, B: 20% -> A: 0%, B: 100%): 50 CV Elution (adjusting the NaCl concentration by adjusting the ratio of buffer A to buffer B until UV baseline is reached) For example, A: 25%, B: 75% = NaCl concentration 500 mM Buffer composition Buffer A: 50 mM sodium phosphate + 2000 mM NaCl + 10 mM EDTA, pH 7.0 Buffer A: 50 mM sodium phosphate + 2000 mM NaCl + 10 mM EDTA, pH 7.0 Buffer B: 50 mM sodium phosphate + 10 mM EDTA, pH 7.0 Buffer B: 50 mM sodium phosphate + 10 mM EDTA, pH 7.0

[0173] That is, the preliminary purified product obtained in Example 2 is mixed with the 2X equilibration buffer shown in Table 21 to prepare the injection sample.

[0174] A CIMmultus™ C4 HLD-4 mL (2 μm) (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and 48 mL of a sample at a concentration of 184.8 μg / mL was injected into the column at a flow rate of 4 mL / min under the conditions shown in Table 21. The column was washed with equilibration buffer until the UV value dropped to baseline. Then, the column was washed with elution buffer under each gradient and stepwise condition, and the eluent fractions were collected (each fraction volume: e.g., 1 mL). The total mRNA and dsRNA concentrations in each fraction were measured according to Experimental Methods 1 and 2.

[0175] The results are shown in Tables 22 and 23 and Figure 7. This confirms that in the gradient elution in Table 22, the dsRNA / mRNA ratio in the first elution fraction was lower than that in the first sample, but the dsRNA / mRNA ratio in the second elution fraction was more similar to that in the first sample. Overall, the distribution of mRNA and dsRNA in the second elution fraction was basically similar. This confirms that if only the first fraction is obtained, the mRNA recovery rate is very low; a certain amount of dsRNA must be obtained to achieve a certain level of mRNA recovery. As shown in Table 23, to achieve an mRNA recovery rate of 80% or more, step elution tests were performed. The results confirmed that at a NaCl concentration of 500 mM, the mRNA recovery rate was 83%, and the dsRNA removal rate was 47%.

[0176] [Table 22]

[0177] [Table 23] Step-by-step washing Total mRNA (μg) mRNA recovery rate Total dsRNA (ng) dsRNA removal rate dsRNA / mRNA ratio C4-HDL sample application 8,870 N / A 2,652 N / A 0.030% C4-HDL elution 1 (NaCl concentration: 500 mM) 7,332 83% 1,417 47% 0.019% C4-HDL elution 2 (NaCl concentration: 300 mM) 973 11% 339 N / A 0.035%

[0178] The above results were obtained when the dsRNA / mRNA ratio was 0.03%, and a spiked test was performed to verify whether a similar removal rate could be ensured when the dsRNA / mRNA ratio was higher. In the spiked test, the dsRNA / mRNA ratio was set to 0.241%, which is about 8 times higher than 0.03% in the above test. Furthermore, since the elution pattern was expected to change with the increase of dsRNA amount, the salt concentration of the first elution buffer was changed.

[0179] The results are shown in Table 24 below, confirming that at a NaCl concentration of 600 mM, the mRNA recovery rate was 70% and the dsRNA removal rate was 40%. It was confirmed that the dsRNA removal rate decreased with increasing mRNA recovery rate, indicating that the dsRNA removal rate decreased with increasing dsRNA / mRNA ratio.

[0180] [Table 24] Stepwise elution (spiking) Total mRNA (μg) mRNA recovery rate Total dsRNA (ng) dsRNA removal rate dsRNA / mRNA ratio C4-HDL sample application 2,167 N / A 5,230 N / A 0.241% C4-HDL elution 1 (NaCl concentration: 600 mM) 1,511 70% 3,139 40% 0.208% C4-HDL elution 2 (NaCl concentration: 500 mM) 357 16% 962 N / A 0.269% C4-HDL elution 3 (NaCl concentration: 300 mM) 72 3% 216 N / A 0.300% C4-HDL elution 4 (NaCl concentration: 0 mM) 151 7% 281 N / A 0.186%

[0181] 5-2. Secondary purification and effect evaluation using Benzyl Ultra resin

[0182] The secondary purification process using Benzyl Ultra resin is summarized in Table 25 below.

[0183] [Table 25] parameter HIC resin (strong) Benzyl Ultra Resin type beads Flow rate 1.67 mL / min (200 cm / hr) Column volume 5 mL Elution type gradient Number of runs 1 2 (Elution flow rate 1 mL / min, gradient decreasing, time extended) Process steps (buffer solution mixing ratio): Volume EQ (A: 100%): 10 CV EQ (A: 100%): 10 CV Re-EQ (A: 100%): until UV baseline Re-EQ (A: 100%): until UV baseline Elution (A: 100%, B: 0% -> A: 0%, B: 100%): 50 CV Elution (A: 50%, B: 50% -> A: 0%, B: 100%): 40 CV Buffer composition Buffer A: 25 mM Tris + 2500 mM Ammonium Sulfate + 10 mM EDTA, pH 7.0 Buffer B: 25 mM Tris + 10 mM EDTA, pH 7.0

[0184] That is, the preliminary purified product obtained in Example 2 is mixed with the 2X equilibration buffer shown in Table 25 to prepare the injection sample.

[0185] A GoPure Benzyl Ultra (Thermo Fisher, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA). Under the conditions shown in Table 25, 20 mL of a 249 μg / mL sample was injected into the column at a flow rate of 1.67 mL / min (Run 1) or 1.1 mL / min (Run 2). The column was then washed with equilibration buffer until the UV value dropped to baseline. Elution buffer was then injected under gradient conditions, and the elution fractions were collected (each fraction volume: e.g., 1 mL). The total mRNA and dsRNA concentrations in each fraction were measured using Experimental Methods 1 and 2.

[0186] The results are shown in Tables 26 and 27 and Figure 8 below, confirming that, unlike Example 5-1, the dsRNA / mRNA ratio of the fraction was higher in Run 1 described in Table 26, indicating a certain degree of separation between dsRNA and mRNA. However, since the mRNA recovery rate of the fraction was confirmed to be 59% to ensure a similar dsRNA removal rate as C4-HLD (43%), Run 2 was performed to check whether additional separation between dsRNA and mRNA could be achieved by reducing the elution gradient and increasing the elution time.

[0187] The results of Run 2 shown in Table 27 below confirm that, while ensuring a similar mRNA recovery rate (61%) as in Run 1, the dsRNA removal rate was 56%, indicating that additional dsRNA can be removed by changing the elution method.

[0188] [Table 26]

[0189] [Table 27]

[0190] 5-3. Secondary purification using Capto Phenyl resin and its effect evaluation

[0191] The secondary purification process using Capto Phenyl resin is summarized in Table 28 below.

[0192] [Table 28] parameter HIC resin (strong) Capto Phenyl (High Substitution) Resin type beads Flow rate 1.67 mL / min (200 cm / hr) Elution flow rate: 1 mL / min Column volume 4.7 mL Elution type gradient Number of runs 1 Process steps (buffer solution mixing ratio): Volume EQ (A: 100%): 10 CV Re-EQ (A: 100%): until UV baseline Elution (A: 100%, B: 0% -> A: 0%, B: 100%): 50 CV Buffer composition Buffer A: 25 mM Tris + 2500 mM Ammonium Sulfate + 10 mM EDTA, pH 7.0 Buffer B: 25 mM Tris + 10 mM EDTA, pH 7.0

[0193] That is, the preliminary purified product obtained in Example 2 is mixed with the 2X equilibration buffer shown in Table 28 above to prepare the injection sample.

[0194] A Hiscrease Capto Phenyl (highly substituted) (Cytiva, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA) column, and 21 mL of a sample at 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. The column was then washed with equilibration buffer until the measured UV value dropped to baseline. Elution buffer was then injected under gradient conditions to elute fractions (volume of each fraction: e.g., 1 mL each). The total mRNA and dsRNA concentrations in each fraction were measured according to Experimental Methods 1 and 2.

[0195] The results are shown in Table 29 and Figure 9 below, confirming that the dsRNA removal rate was 50% and the mRNA yield was 81%. In other words, Capto Phenyl resin exhibited the best performance compared to C4-HLD and Benzyl Ultra.

[0196] [Table 29]

[0197] The results of Examples 5-1 to 5-3 are summarized in Table 30 below.

[0198] [Table 30]

[0199] That is, it was confirmed that although there are differences in dsRNA removal rates among HIC resins, the monolithic column used in Example 5-1 had an mRNA recovery rate of about 80%, a dsRNA removal rate of about 50%, and the shortest process time (shortened to about 1 / 3).

[0200] Example 6. mRNA production, purification, and sample preparation to evaluate the innate immune response and toxicity induced by the IVT product purification process at different dsRNA concentrations.

[0201] Five samples with different dsRNA concentrations were prepared, and in vivo innate immune response and toxicity were evaluated for each sample. To assess the effects of different concentrations of dsRNA, mRNA was synthesized and purified using the process shown in Figure 10.

[0202] 6-1. In vitro transcription

[0203] In vitro transcription is the process of synthesizing mRNA.

[0204] The prepared template DNA was reacted with T7 RNA polymerase, buffer, NTPs (including natural and chemically modified NTPs) and other necessary elements for IVT at 37ºC for 4 hours, as shown in Table 31 below.

[0205] [Table 31] reagents IVT final concentration supplier Catalog Number water* - - - ATP 10 mM Roche 04980824103 CTP 10 mM Roche 04980875103 GTP 10 mM Roche 04980859103 m1ΨTP 10 mM Thermo R0491SKB012 CleanCap AG (3'OMe) 8.0 mM TriLink N-7413 reaction buffer 1X self made - Template DNA 40 μg / mL self made - RI 1 kU / mL Roche 09537589103 PPase 2 U / mL Roche 08140677103 T7 RNAP 24 kU / mL Thermo EP011SKB011 *All reagents were added based on the total IVT reaction volume for each reaction, with water added to reach the final volume.

[0206] After the reaction was complete, the DNA was treated with 50 U / mL DNase I (03539121103, Roche, USA), then reacted at 37ºC for 30 minutes to remove the template DNA. The DNA was then treated with 70 mM EDTA and incubated for 3 minutes. 5X volumes of RNase-free water were added and the mixture was centrifuged. The resulting IVT product was used for subsequent purification processes.

[0207] 6-2. Preliminary purification using affinity chromatography

[0208] The IVT product obtained in Example 6-1 was initially purified using a column containing oligo(A) tail bound to oligo(dT) by the method described in Example 2.

[0209] The specific methods are summarized in Table 32 below.

[0210] [Table 32] Process parameters condition resin Oligomeric dT18 (C12 adapter) 40 mL monolithic column Column volumetric volume (mL) 40 Sample quality (mg / mL resin) Approximately 2.00 Temperature (ºC) room temperature Buffer composition EQ 10 CV 10 mM phosphate + 250 mM NaCl + 5 mM EDTA (pH 6.8) Sample N / A N / A EQ again 10 CV 10 mM phosphate + 250 mM NaCl + 5 mM EDTA (pH 6.8) washing 40 CV 50 mM phosphate + 5 mM EDTA (pH 6.8) Washout 6 CV 5 mM Tris (pH 7.0) CIP 30 CV (0.5 N NaOH) Washout 10 CV 5 mM Tris (pH 7.0) store 6 CV 20% EtOH

[0211] Specifically, an 8 mL (2 µm) CIMmultus oligo-dT (C12 adapter) column (Sartorius, USA) was mounted on an AKTA Avant 150 (Cytiva, USA) column, and the 2.0 mg / mL IVT product of Example 6-1, mixed with 3 mL of sample preparation buffer (20 mM sodium phosphate + 500 mM NaCl + 10 mM EDTA, pH 6.6), was loaded into the column. The column was then reequilibrated with 10 CV of equilibration buffer, and the column containing the bound product was washed with 40 CV or less of wash buffer. Then, 6 CV of elution buffer was loaded to recover the IVT product containing the poly(A) tail bound to the column. The column was then washed with CIP buffer.

[0212] The obtained mRNA purification solution was concentrated to 2.5 mg / mL using Amicon Ultra-15 30 KD (UFC903024, MILLIPORE, USA) at 3,000 g and 6ºC, and then buffer exchanged (1 mM sodium citrate + 200 mM NaCl (pH 5.7)).

[0213] 6-3. Secondary purification using size exclusion chromatography

[0214] 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 the dsRNA concentration was high in the pre-peak portion during the SEC process. Therefore, after performing the SEC process using the method described in Table 33 below, the flow-through peaks were collected. The pre-peak with higher dsRNA concentration was named high dsRNA, and the post-peak with lower dsRNA concentration was named low dsRNA.

[0215] For example, for high dsRNA, use flow-through fractions 1-5 from Example 3; for low dsRNA, use fractions 18-27 from Example 3.

[0216] The specific methods are summarized in Table 33 below.

[0217] [Table 33] Process parameters condition column Superdex 200 PG Column volume (mL) 318.4 Sample concentration (mg / mL) 2.7 Sample loading volume (mL) 4.4 Sample quality (mg) 11.6 Flow rate (mL / min) 2.6 Buffer composition EQ 2 CV 1 mM sodium citrate + 200 mM NaCl (pH 5.7) Sample N / A N / A EQ again 1.5 CV 1 mM sodium citrate + 200 mM NaCl (pH 5.7)

[0218] The obtained mRNA purification solutions (for each collected sample) were concentrated to 0.7 mg / mL or more using an Amicon Ultra-15 30 KD (UFC903024, MILLIPORE, USA) at 3,000 g and 6ºC, and then buffer-exchanged with 1 mM sodium citrate (pH 6.4).

[0219] 6-4. LNP Packaging and Sample Preparation

[0220] Each of the obtained high-dsRNA and low-dsRNA was encapsulated using ALC-0315 LNP. The final concentration was set at 0.2 mg / mL. Samples were aliquoted, frozen, thawed, and used on the same day as in vivo assessments of innate immune response and toxicity.

[0221] The dsRNA concentrations of the high-dsRNA and low-dsRNA samples obtained 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 primary experimental samples for innate immune response and toxicity test samples were prepared at the concentrations described in Table 35 below.

[0222] [Table 34] # sample Mixing ratio (%) dsRNA / mRNA (%) High dsRNA low dsRNA 1 High dsRNA 100 0 0.06 2 medium dsRNA 50 50 0.03 3 low dsRNA 0 100 0.005

[0223] [Table 35] # sample Mixing ratio dsRNA / mRNA (%) High dsRNA low dsRNA 1 High dsRNA 100 0 0.04 2 Medium 1 dsRNA 75 25 0.03 3 Medium 2 dsRNA 50 50 0.02 4 Medium 3 dsRNA 25 75 0.01 5 low dsRNA 0 100 0.002

[0224] Example 7. Experimental method for evaluating innate immune response and toxicity under varying dsRNA concentrations based on IVT product purification process.

[0225] 7-1. Animal Experiments

[0226] 7-1-1. Innate Immune Response

[0227] Five-week-old female Balb / C and C57BL / 6 mice (Orient Bio, female) were purchased and, after a week of acclimatization, were used for experiments at 6 weeks of age. Samples were prepared according to the experimental plan, and 50 to 200 µL of insulin was administered intramuscularly to the right thigh of each mouse using a 33-gauge insulin syringe (0.5 mL). Then, 500 µL or more of blood was collected from each mouse via whole blood collection.

[0228] To facilitate serum separation, allow the collected blood to stand at room temperature for 30 minutes or longer to allow it to coagulate, then centrifuge (at 10,000 rpm and 4ºC for 10 minutes) and transfer the supernatant to a new 1.5 mL tube. To separate pure serum, centrifuge the blood again (at 10,000 rpm and 4ºC for 10 minutes), transfer the supernatant to a new 1.5 mL tube, and store at -20ºC until analysis.

[0229] 7-1-2. Toxicity

[0230] Six-week-old female Balb / C mice (Orient Bio, female) were purchased and, after a week of acclimatization, were used for experiments at 7 weeks of age. Samples were prepared according to the experimental plan and collected using an insulin syringe (size 33, 0.5 mL), with 200 µL administered intravenously to each mouse via the tail vein. Then, 500 µL or more of blood was collected from each mouse via whole blood collection.

[0231] To facilitate serum separation, allow the collected blood to stand at room temperature for 30 minutes or longer to coagulate, then centrifuge (at 10,000 rpm and 4ºC for 10 minutes) and transfer the supernatant to a new 1.5 mL tube. To separate pure serum, centrifuge the blood again (at 10,000 rpm and 4ºC for 10 minutes), transfer the supernatant to a new 1.5 mL tube, and store at -20ºC until analysis.

[0232] 7-2. Assess innate immune response by measuring IFN-α, IFN-β, IP-10 and MCP-1 using a multianalytical assay.

[0233] To measure IFN-α, IP-10, and MCP-1, the LEGENDplex mouse antiviral response combination (13-fold) with filter plate kit (Biolegend, 740621) was used. The assay was performed according to the kit instructions.

[0234] First, take one vial of lyophilized standards from the LEGENDplex kit and reconstitute it in 250 μL of LEGENDplex assay buffer for 10 minutes at room temperature. Then, perform a 4-fold serial dilution starting with the highest concentration of standard to prepare a total of 8 standard aliquots (including buffer only) in 1.7 mL tubes. Additionally, prepare a sample by diluting 25 μL of serum obtained in Example 7-1 by a 2-fold dilution with 25 μL of LEGENDplex assay buffer.

[0235] Premixed beads were prepared to account for the number of standards and samples to be analyzed. 25 μL of premixed beads were used for each analytical sample. The premixed beads included in the kit were vortexed for 1 minute or longer. 25 mL of 20X LEGENDplex wash buffer was diluted with 475 mL of distilled water and stored at 4ºC before use. 5 mL of LEGENDplex assay buffer was added to the vial containing lyophilized matrix A and reconstituted over 15 minutes.

[0236] Use the filter plate included in the kit. Add 100 μL of LEGENDplex wash buffer to each well of the filter plate and incubate at room temperature for 1 minute. Remove any LEGENDplex wash buffer from the wells using a vacuum manifold, then add 25 μL of matrix A and 25 μL of standard to each well, followed by 25 μL of the analytical sample and 25 μL of assay buffer included in the kit. Next, vortex the premixed beads for 30 seconds, then add 25 μL of beads to each well. To prevent bead settling, mix the beads intermittently. For the reaction, shake the plate at 500 rpm at room temperature for 2 hours using a plate shaker.

[0237] After the reaction is complete, place the plate on a vacuum manifold and drain the liquid under vacuum by aspiration. Wash all wells with 200 μL of LEGENDplex wash buffer, repeating this process twice. Then, add 25 μL of the detection antibody included in the kit to each well. Next, for the reaction, shake the plate at 500 rpm at room temperature for 1 hour using a plate shaker. During shaking, wrap the plate with aluminum foil to block light.

[0238] After the reaction was complete, the reaction solution was removed from the wells using a vacuum manifold, and then all wells were washed twice 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 a 1.1 mL tube.

[0239] Samples were analyzed 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).

[0240] Statistical analysis was performed using Graphpad Prism 10. Normality was tested using the D'Agostino & Pearson test. If the distribution was normal, a simple one-way ANOVA was used; otherwise, the Kruskal-Wallis test was used. Post-hoc analysis was performed using the Turkey test (p < 0.05, *; p < 0.005, **; p < 0.0005, ***; p < 0.0001, ****).

[0241] 7-3. Assess innate immune response by measuring IFN-α using ELISA.

[0242] To measure serum IFN-α, a mouse IFN-α ELISA kit (BMS6027, Invitrogen, USA) was used. The assay was performed according to the kit instructions.

[0243] If wash buffer crystals appear, dissolve the wash buffer completely at room temperature, then dilute 50 mL of the wash buffer concentrate with 950 mL of distilled water. If assay buffer crystals appear, dissolve the assay buffer completely at room temperature, then dilute 10 mL of the assay buffer concentrate with 190 mL of distilled water. Dissolve one vial of mouse IFN-α standard completely in the specified amount of distilled water, then serially dilute the 4,000 pg / mL standard stock solution seven times with calibration diluent (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 with assay buffer (120 μL biotin conjugate + 11.88 mL assay buffer (1x)); and streptavidin-HRP was diluted 1 / 300 with assay buffer (40 μL streptavidin-HRP + 11.96 mL assay buffer (1x)).

[0244] Wash the plate provided in the kit twice with 400 μl of wash buffer. Then, add 50 μl of assay buffer, 50 μl of 1 / 1.5 diluted serum, and 50 μl of prepared standard to each well. Add 50 μl of calibrator dilution to each blank well. Cover the plate with the plate sealer and incubate at 650 rpm and room temperature for 1 hour. Then, invert the plate to discard the solution and wash each well four times with 300 μl of wash buffer, gently tapping the plate through several layers of paper towels to completely remove any residual solution. Then, add 100 μl of diluted streptavidin-HRP to each well and incubate the plate at 450 rpm and room temperature for 1 hour. Then, invert the plate to discard the solution and wash each well four times with 400 μl of wash buffer, gently tapping the plate through several layers of paper towels to completely remove any residual solution. Then, add 100 μl of TMB solution to each well and incubate the plate at room temperature for 30 minutes. At this point, wrap the plate with foil to block light. Add 100 μL of stop solution to each well to terminate the reaction, and measure the absorbance at 450 nm and 620 nm using a microplate reader within 5 minutes. Then correct the absorbance by passing the OD at 450 nm through the OD at 620 nm.

[0245] The experimental results were analyzed using the SoftMax Pro program. Specifically, a standard curve was plotted using 5 parameters, 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.

[0246] The D'Agostino & Pearson test was used to test for normality. If the distribution is normal, a standard one-way ANOVA was used; if the distribution is not normal, the Kruskal-Wallis test was used. Post-hoc analysis was performed using the Turkey test (p < 0.05, *; p < 0.005, **; p < 0.0005, ***; p < 0.0001, ****).

[0247] 7-4. Assess innate immune response by measuring IFN-β using ELISA.

[0248] To measure serum IFN-β, a high-sensitivity mouse IFN-β ELISA kit (42410-1, PBL, USA) was used. The assay was performed according to the kit instructions.

[0249] Take 60 μL of the 1,000 pg / mL standard working stock solution and dilute it in 940 μL of sample diluent (60 pg / mL). Perform a 1 / 2 serial dilution to prepare 7 dilutions (60, 30, 15, 7.5, 3.75, 1.87, and 0.94 pg / mL). Thaw the serum obtained in Example 7-1 at room temperature and then perform a 1 / 3 dilution with the sample diluent (40 μL sample diluent + 20 μL serum). The antibody solution was prepared by diluting it 1:60 with the antibody diluent 15 minutes before use, and the HRP solution was prepared by diluting it 1:70 with the HRP diluent 15 minutes before use. Prepare the wash buffer by adding 50 mL of the concentrated wash solution containing completely dissolved crystals to a bottle containing 450 mL of distilled water and mixing.

[0250] Add 50 μL of serum buffer, 50 μL of diluted serum, and 50 μL of prepared standard to each well. Add 50 μL of sample diluent to each blank well. Cover the plate with plate sealer and incubate at 650 rpm and room temperature for 1 hour. Then invert the plate to discard the solution and wash each well four times with 300 μL of wash buffer, gently tapping the plate through several layers of paper towels to completely remove any residual solution. Next, add 50 μL of prepared antibody solution to each well and incubate the plate at 650 rpm and room temperature for 30 minutes. Then invert the plate to discard the solution and wash each well four times with 300 μL of wash buffer, gently tapping the plate through several layers of paper towels to completely remove any residual solution. Finally, add 50 μL of prepared HRP solution to each well and incubate the plate at 650 rpm and room temperature for 10 minutes. The plate was then inverted to discard the solution, and each well was washed four times with 300 μL of wash buffer each time. Finally, the plate was gently patted through several layers of paper towels to completely remove any residual solution. Next, 100 μL of TMB solution was added to each well, and the plate was incubated at room temperature for 10 minutes. At this point, the plate was wrapped with foil to block light. Then, 100 μL of stop solution was added to each well to terminate the reaction, and the absorbance was measured at 450 nm using a ELISA reader (Molecular Devices, Spectramax M3) within 5 minutes.

[0251] The experimental results were analyzed using the SoftMax Pro program. Specifically, a standard curve was plotted using 4 parameters, 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.

[0252] Statistical analysis was performed using GraphPad Prism 10. Normality was tested using the D'Agostino & Pearson test. If the distribution was normal, a standard one-way ANOVA was used; otherwise, the Kruskal-Wallis test was used. Post-hoc analysis used the Turkey test (p < 0.05, *; p < 0.005, **; p < 0.0005, ***; p < 0.0001, ****).

[0253] 7-5. Measurement of ALT and AST in cytotoxicity assessment

[0254] Serum ALT and AST levels were measured by Chaon Corporation (Korea) using an AU680 clinical chemistry analyzer (Beckman Coulter, USA).

[0255] Example 8. Preliminary experiment to evaluate the innate immune response under varying dsRNA concentrations based on the IVT product purification process.

[0256] Since the innate immune response dependent on dsRNA concentration had not been previously evaluated, preliminary experiments were conducted as shown in Table 36 below to select appropriate dsRNA concentrations, blood collection time points, and mouse strains for comparing type I IFN and chemokine secretion levels.

[0257] [Table 36] serial number Group strain injection animal numbers mRNA (μg) Volume (μL) 1 High dsRNA Balb / c 10 μg (High 10) 50 12 2 C57BL / 6 10 μg (High 10) 50 12 3 medium dsRNA Balb / c 10 μg (High 5 + Low 5) 50 12 4 C57BL / 6 10 μg (High 5 + Low 5) 50 12 5 low dsRNA Balb / c 10 μg (low 10) 50 12 6 C57BL / 6 10 μg (low 10) 50 12 7 PBS Balb / c N / A 50 12 8 C57BL / 6 N / A 50 12

[0258] Blood was collected at 1 hour, 3 hours, and 6 hours after drug 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. IFN-α, IFN-β, IP-10, and MCP-1 were analyzed using the methods of Examples 7-2 to 7-4.

[0259] The results are shown in Figure 11. When IFN-α concentration was analyzed by ELISA, IFN-α was not detected at 1 hour and 3 hours after administration, regardless of dsRNA concentration, and was only detected at 6 hours. Furthermore, it was confirmed that the secretion level of IFN-α increased with increasing dsRNA concentration, and in the medium dsRNA group (dsRNA / mRNA (%) = 0.03), the IFN-α secretion level of the C57BL / 6 strain was higher than that of the Balb / C strain.

[0260] In addition, to simultaneously confirm the detection of IFN-α, IFN-β, IP-10 and MCP-1, a multianalytical assay was performed using the method of Examples 7-2. Due to the limited availability of serum samples, serum from the Balb / C strain (6 hours post-immunization) was used for analysis, rather than the C57BL / 6 strain which secretes high levels of IFN-α.

[0261] The results are shown in Figure 12, which confirm that the secretion level of IFN-α increases with the increase of dsRNA concentration, and IFN-α, IP-10 and MCP-1 are detected at all dsRNA concentrations except IFN-β.

[0262] In addition, in order to check whether IFN-β was still secreted even in samples with lower dsRNA concentrations than high dsRNA samples, serum from the C57BL / 6 strain (6 hours post-immunization) was analyzed using the method in Examples 7-3 with an IFN-β ELISA kit that has a low detection limit.

[0263] The results are shown in Figure 13, which confirm that IFN-β, which was not detected in the multianalytical assay, can be detected by the IFN-β ELISA kit with a low detection limit. The results are similar to the correlation between dsRNA concentration and IFN-α secretion level shown in Figures 11 and 12, where the secretion level of IFN-β increases with increasing dsRNA concentration.

[0264] Based on the above results, the blood collection time for this experiment was selected as 6 hours after immunization, at which time IFN-α could be detected. The mouse strain selected was the C57BL / 6 strain, which could detect higher IFN-α at low dsRNA concentrations. In addition, since the lowest dsRNA concentration that can identify IFN-α and IFN-β secretion is 0.03% (dsRNA / mRNA (%)), corresponding to the medium dsRNA group, the dsRNA concentration of the high dsRNA group in this experiment was set at 0.04% (dsRNA / mRNA (%)).

[0265] Example 9. Evaluation of innate immune response under varying dsRNA concentrations based on IVT product purification process.

[0266] This experiment used the mouse strain and blood collection time points determined in Example 8, as shown in Table 37 below. Serum was obtained using the method of Example 7-1, and the secretion levels of IFN-α, IFN-β, IP-10, and MCP-1 in the obtained serum based on dsRNA concentration were analyzed using the methods of Examples 7-2 and 7-4. That is, the secretion levels of IFN-α, IP-10, and MCP-1 were analyzed using the method of Example 7-2, and the secretion level of IFN-β was analyzed using a low-detection-limit ELISA kit using the method of Example 7-4.

[0267] [Table 37] serial number Group strain injection animal numbers mRNA (μg) Volume (μL) 1 High dsRNA C57BL / 6 10 μg (High 10) 50 8 2 Medium 1 dsRNA 10 μg (High 7.5 + Low 2.5) 50 8 3 Medium 2 dsRNA 10 μg (High 5 + Low 5) 50 8 4 Medium 3 dsRNA 10 μg (High 2.5 + Low 7.5) 50 8 5 low dsRNA 10 μg (low 10) 50 8 6 PBS N / A 50 8

[0268] The results are shown in Figure 14, which confirm that the secretion levels of IFN-α, IFN-β, IP-10 and MCP-1 all showed an increasing trend with the increase of dsRNA concentration. Moreover, there were no statistically significant differences between the low dsRNA group (dsRNA / mRNA (%) = 0.002) and the medium dsRNA group (dsRNA / mRNA (%) = 0.01) and the control PBS group in all evaluation items.

[0269] Therefore, it was confirmed that a dsRNA concentration of 0.02% (dsRNA / mRNA (%)) or higher, corresponding to the intermediate 2 dsRNA group, is a dsRNA concentration that can induce an innate immune response.

[0270] That is, it has been confirmed that the mRNA purified by primary affinity chromatography and secondary SEC process according to the present invention has a low dsRNA concentration and does not induce or significantly reduce the innate immune response.

[0271] Example 9. Evaluation of in vivo toxicity under varying dsRNA concentrations based on IVT product purification process.

[0272] Considering the biodistribution of mRNA / LNP after systemic administration, the liver is the main organ where mRNA / LNP is distributed. Therefore, the liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were selected as toxicity assessment 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 experiments were conducted at the dsRNA concentrations shown in Table 35 above.

[0273] As shown in Table 38 below, the experiment was conducted in 6 groups, including a control group. Blood was collected 24 hours after drug administration, which was the time point at which acute hepatotoxicity was observed. AST and ALT, based on dsRNA concentration, were analyzed in serum obtained using the method of Example 7-1 according to the method of Example 7-5.

[0274] [Table 38] serial number Group strain injection animal numbers mRNA (μg) Volume (μL) 1 High dsRNA Balb / c 100 μg (High 100) 200 3 2 Medium 1 dsRNA 100 μg (High 75 + Low 25) 200 3 3 Medium 2 dsRNA 100 μg (High 50 + Low 50) 200 3 4 Medium 3 dsRNA 100 μg (High 25 + Low 75) 200 3 5 low dsRNA 100 μg (low 10) 200 3 6 Vector (empty LNP) N / A 200 3

[0275] Statistical analysis of toxicity assessment results based on dsRNA concentration was performed using GraphPad Prism 10. One-way ANOVA was used, and post-hoc analysis was performed using the Turkey test.

[0276] The results, as shown in Figure 15, confirmed that the secretion levels of ALT and AST both increased with increasing dsRNA concentration. Furthermore, statistical analysis confirmed that only the high dsRNA group (dsRNA / mRNA (%) = 0.04) showed significant differences in ALT and AST levels compared to other groups. Except for the high dsRNA group, the other groups did not show significant differences compared to the control vector (empty LNP) group.

[0277] Thus, it was confirmed that although toxicity may vary depending on GOI, mouse strains and other species, as well as the LNP, dosage and method used, no toxicity due to dsRNA was observed up to 0.03% (dsRNA / mRNA (%)) corresponding to the intermediate 1 dsRNA group.

[0278] That is, it was confirmed that the mRNA purified by the first affinity chromatography and the second SEC process according to the present invention had a low dsRNA concentration and did not show or significantly reduce in vivo toxicity.

[0279] Example 10. Optimization of salt concentration in sample preparation buffer and equilibration buffer after preliminary purification by affinity chromatography.

[0280] The binding of oligomeric dT resin to mRNA occurs via hydrogen bonding, as NaCl masks the repulsive force between the (-)phosphate groups of the resin ligands and the (-)phosphate groups 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 may lead to aggregation; therefore, it is necessary to select an appropriate NaCl concentration in the EQ buffer before checking the DBC. Thus, we examined the mRNA productivity and quality of each construct as a function of NaCl concentration to determine the appropriate process range.

[0281] IVT was performed as in Example 1 to obtain mRNA of genes 1, 2 and 3. Equilibration buffer was prepared according to the conditions in Table 39. The turbidity of oligodT loading was checked under each condition. Each obtained product was subjected to affinity chromatography twice as shown in Table 40. The eluent was then analyzed.

[0282] Turbidity was checked by measuring UV at 350 nm using a UV spectrophotometer (Libra S50), and the amount and yield of eluent were analyzed as in Experimental Method 1.

[0283] [Table 39] Operation number buffer solution Buffer solution concentration in moles (mM) pH NaCl molar concentration (mM) EDTA molar concentration (mM) 1 phosphate 10 6.7 300 5 2 400 3 500 4 600 5 700 6 800 7 900 8 1000

[0284] [Table 40] Process parameters condition resin Oligomeric dT18 (C12 connector) 0.2 mL monolithic 96-well plate Column volumetric volume (mL) 0.2 Sample quality (mg / mL resin) 1.0 Temperature (°C) room temperature Buffer composition EQ 10 CV Table 39 Sample N / A N / A EQ again 10 CV Table 39 washing 40 CV 50 mM phosphate + 5 mM EDTA (pH 6.7) Washout 6 CV 5 mM tris (pH 7.0) CIP 30 CV (0.5 N NaOH) Washout 10 CV 5 mM tris (pH 7.0) store 6 CV 20% EtOH

[0285] Specifically, a 0.2 mL CIMmultus oligo-dT (C12 adapter) 96-well plate (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA). A 1.0 mg / mL IVT product, mixed with sample preparation buffer (20 mM sodium phosphate + 2X concentration of NaCl from Table 39 + 10 mM EDTA, pH 6.5), was loaded into the column. The column was then reequilibrated with 10 CV of equilibration buffer, and the column containing the bound product was washed with 40 CV or less of wash buffer. A 6 CV elution buffer was then loaded to recover the IVT product containing the poly(A) tail bound to the column. The column was then washed with 30 CV of injected CIP buffer and stored in 20% ethanol.

[0286] The results are shown in Tables 41 to 43. Turbidity in all constructs increased with increasing NaCl concentration, although the degree of turbidity increase varied among different constructs.

[0287] [Table 41] Turbidity results of NaCl molar concentration study in loading / EQ buffer (Gene 1) EQ buffer 0 h 1 h 2 h 3 h NaCl (mM) 300 0.008 0.008 0.010 0.011 400 0.011 0.011 0.011 0.013 500 0.013 0.015 0.017 0.020 600 0.012 0.028 0.052 0.167 700 0.015 0.134 0.331 0.476 800 0.02 0.39 0.555 0.64 900 0.022 0.563 0.695 0.754 1000 0.026 0.675 0.79 0.852 EQ buffer 0 h 1 h 2 h 3 h NaCl (mM) 300 0.008 0.008 0.010 0.011 400 0.011 0.011 0.011 0.013 500 0.013 0.015 0.017 0.020 600 0.012 0.028 0.052 0.167 700 0.015 0.134 0.331 0.476 800 0.02 0.39 0.555 0.64 900 0.022 0.563 0.695 0.754 1000 0.026 0.675 0.79 0.852

[0288] [Table 42] Turbidity results of NaCl molar concentration study in loading / EQ buffer (Gene 2) EQ buffer 0 h 1 h 2 h 3 h NaCl (mM) 300 0.018 0.023 0.025 0.030 400 0.024 0.033 0.042 0.057 500 0.026 0.071 0.109 0.194 600 0.027 0.017 0.291 0.485 700 0.031 0.345 0.539 0.7 800 0.036 0.577 0.727 0.827 900 0.054 0.74 0.841 0.914 1000 0.1 0.823 0.9 0.964

[0289] [Table 43] Turbidity results of NaCl molar concentration study in loading / EQ buffer (Gene 3) EQ buffer 0 h 1 h 2 h 3 h NaCl (mM) 300 0.021 0.022 0.023 0.020 400 0.020 0.021 0.025 0.024 500 0.023 0.026 0.028 0.030 600 0.023 0.029 0.035 0.039 700 0.026 0.035 0.045 0.057 800 0.027 0.047 0.064 0.097 900 0.031 0.068 0.102 0.183 1000 0.03 0.098 0.16 0.269

[0290] Furthermore, for gene 1, as shown in Tables 41, 44, and 45, oligomeric dT loading began to precipitate at 600 mM, making the process impossible. However, the process could proceed at 300-500 mM, with %CVs between yields within 2%, %CVs between purities within 1%, and %CVs between dsRNA contents of 10.20%, confirming no difference in QA / PA within the processable range. In other words, all QA / PA acceptance criteria were met at 300-500 mM: yield (%) ≥ 80.0, dsRNA content (ng / mg) ≤ 1000, and purity (%) ≥ 80.0.

[0291] [Table 44] Yield results of NaCl molar concentration study in loading / EQ buffer (Gene 1) EQ buffer Elution volume (ug) Yield (%) CV (%) First run Second run First run Second run average NaCl (mM) 300 214.80 83.49 107.22 101.94 104.58 0.87 400 N / A 209.99 N / A 104.82 104.82 500 213.96 211.80 106.80 105.72 106.26

[0292] [Table 45] Results of purity and dsRNA content studies of NaCl molar concentration in loading / EQ buffer (gene 1) EQ buffer purity(%) CV (%) dsRNA content (ng / mg) CV (%) First run Second run average First run Second run average NaCl (mM) 300 93.45 89.24 91.35 0.92 1.87 1.19 1.53 10.20 400 N / A 92.65 92.65 N / A 1.76 1.76 500 92.82 93.06 92.94 N / A 1.88 1.88

[0293] In the case of gene 2, as shown in Tables 42, 46, and 47, oligomeric dT precipitation began at 700 mM, with highly visible turbidity observed at 500 mM and 600 mM, indicating column blockage during actual process runs. At 300–400 mM, under operable process conditions, the %CV between yields was within 2%, the %CV between purities was within 1%, and the %CV between dsRNA contents was 7.17%, confirming no difference in QA / PA within the operable range. That is, all QA / PA acceptance criteria were met at 300–400 mM: yield (%) ≥ 80.0, dsRNA content (ng / mg) ≤ 1000, and purity (%) ≥ 80.0.

[0294] [Table 46] Yield results of NaCl molar concentration study in loading / EQ buffer (Gene 2) EQ buffer Elution volume (ug) Yield (%) CV (%) First run Second run First run Second run average NaCl (mM) 300 244.13 254.10 121.78 126.75 124.26 1.13 400 255.67 250.61 127.53 125.01 126.27

[0295] [Table 47] Results of purity and dsRNA content studies in the NaCl molar concentration study of loading / EQ buffer (gene 2) EQ buffer purity(%) CV (%) dsRNA content (ng / mg) CV (%) First run Second run average First run Second run average NaCl (mM) 300 90.99 91.06 91.03 0.10 1.11 0.97 1.04 7.17 400 90.98 90.80 90.89 N / A 1.15 1.15

[0296] Furthermore, for gene 3, as shown in Tables 43, 48, and 49, no precipitation occurred upon loading of oligomeric dT, but column blockage occurred during process runs at 900 mM and 1000 mM. At 300–800 mM, the %CV between yields was within 2%, the %CV between purities was within 1%, and the %CV between dsRNA contents was 7.26%, confirming no difference in QA / PA within the manageable range. That is, all QA / PA acceptance criteria were met at 300–800 mM: yield (%) ≥ 80.0, dsRNA content (ng / mg) ≤ 1000, and purity (%) ≥ 80.0.

[0297] [Table 48] Yield results of NaCl molar concentration study in loading / EQ buffer (Gene 3) EQ buffer Elution volume (ug) Yield (%) CV (%) First run Second run First run Second run average NaCl (mM) 300 186.91 190.50 93.30 95.09 94.20 1.29 400 191.00 191.53 95.35 95.61 95.48 500 194.58 196.74 97.13 98.21 97.67 600 189.51 195.29 94.60 97.49 96.04 700 195.74 192.99 97.71 96.34 97.02 800 191.17 191.16 95.43 95.42 95.43

[0298] [Table 49] Results of purity and dsRNA content in the NaCl molar concentration study of the loading / EQ buffer (gene 3) EQ buffer Purity (%) CV (%) dsRNA content (ng / mg) CV (%) First run Second run average First run Second run average NaCl (mM) 300 78.47 79.56 79.02 0.64 16.59 18.02 17:30 7.26 400 78.58 79.47 79.03 16.42 16.49 16.45 500 78.48 79.26 78.87 18.68 17.43 18.05 600 78.19 78.60 78.40 15.60 15.62 15.61 700 77.65 78.58 78.12 14.57 16.89 15.73 800 77.71 77.94 77.83 14.19 15.49 14.84

[0299] In summary, it has been confirmed that the usable NaCl concentration in the EQ buffer varies for each construct: 300-500 mM for Gene 1, 300-400 mM for Gene 2, and 300-800 mM for Gene 3. Furthermore, it has been confirmed that a NaCl concentration of 300-400 mM in the EQ buffer is a usable concentration for all constructs.

[0300] Example 11. Evaluation of the composition and pH of the eluent buffer in the preliminary purification using affinity chromatography.

[0301] Since the ligands of oligomeric dT resin and mRNA are hydrogen-bonded under high-salt conditions, a low-salt buffer is used for elution. Therefore, based on the above examples and existing literature, the elution buffer range was selected as shown in Table 50, and the effects of the elution buffer composition and pH on mRNA yield and quality for each construct were examined to determine a suitable process range.

[0302] That is, after performing the chromatography (oligomeric dT) process according to the method in Table 51, the eluent was analyzed according to the method in Example 10. Each condition was run twice. Since the eluent may need to be allowed to stand before proceeding to the next process (i.e., the mRNA UF / DF process), the purity analysis was performed for 4 days at room temperature and 7 days under refrigeration conditions to select a buffer composition and pH that could ensure a certain level of stability.

[0303] [Table 50] Buffer composition used for elution buffer composition and pH studies Operation number buffer solution Buffer solution concentration in moles (mM) pH 1 citrate 5 5.0 2 5.5 3 6.0 4 6.5 5 7.0 6 7.5 7 Tris 7.0 8 UPW N / A N / A 9 Acetate 5 5.0 10 5.5 11 6.0 12 6.5

[0304] [Table 51] Process conditions used for studying the composition and pH of the elution buffer Process parameters condition resin Oligomeric dT18 (C12 connector) 0.2 mL monolithic 96-well plate Column volumetric volume (mL) 0.2 Sample quality (mg / mL resin) 1.2 (Gene 1), 1.4 (Gene 2), 1.1 (Gene 3) Temperature (°C) room temperature Buffer composition EQ 10 CV 10 mM phosphate + 350 mM NaCl + 5 mM EDTA (pH 6.7) Sample N / A N / A EQ again 10 CV 10 mM phosphate + 350 mM NaCl + 5 mM EDTA (pH 6.7) washing 40 CV 50 mM phosphate + 5 mM EDTA (pH 6.7) Washout 6 CV Table 50 CIP 30 CV (0.5 N NaOH) Washout 10 CV Table 50 store 6 CV 20% EtOH

[0305] Specifically, a 0.2 mL CIMmultus oligo-dT (C12 adapter) 96-well plate (Sartorius, USA) column was mounted on an AKTA Avant 150 (Cytiva, USA), and the IVT product mixed with sample preparation buffer (20 mM sodium phosphate + 700 mM NaCl + 10 mM EDTA, pH 6.5) was loaded into the column. The column was then reequilibrated with 10 CV of equilibration buffer, and the column containing the bound product was washed with 40 CV or less of wash buffer. Then, 6 CV of elution buffer was loaded to recover the IVT product containing the poly(A) tail bound to the column. The column was then washed with 30 CV of injected CIP buffer and stored in 20% ethanol.

[0306] Results: For gene 1, as shown in Tables 52 and 53, regardless of the composition and pH of the elution buffer, the %CV between yields was within 2%, and the %CV between purities was within 1%, confirming no difference in QA / PA within the manageable range. The dsRNA content of the elution buffer was examined, confirming a %CV of 30.07% between dsRNA contents, which varied with the composition and pH of the elution buffer. Except for citrate buffer (pH 5.0), lower dsRNA content was confirmed in citrate buffer compared to other buffers. This was due to differences in buffer conductivity, and it was determined that the conductivity of citrate buffer (pH 5.0) exceeded 900 mS / cm, approximately twice that of other buffers, thus failing to elute dsRNA strongly bound to oligomeric dT resin.

[0307] In addition, as shown in Table 53, the storage stability of the eluent in all elution buffers was confirmed to be stable for 4 days and 7 days at room temperature, and the maximum purity %CV was 1.06% between days 0 and 4 at room temperature and between days 0 and 7 at room temperature.

[0308] 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 under refrigeration. However, in order to reduce dsRNA content, citrate buffer with pH 5.5 - 7.5 was determined to be suitable as an elution buffer to reduce dsRNA content.

[0309] [Table 52] Yield results from elution buffer composition and pH studies (Gene 1) Elution buffer Elution volume (ug) Yield (%) CV (%) First run Second run First run Second run average citrate pH 5.0 251.02 235.66 107.69 101.10 104.39 1.89 pH 5.5 244.38 252.05 104.84 108.13 106.48 pH 6.0 250.14 250.08 107.31 107.28 107.30 pH 6.5 243.57 251.31 104.49 107.81 106.15 pH 7.0 238.82 246.68 102.45 105.83 104.14 pH 7.5 237.16 247.05 101.74 105.98 103.86 Tris pH 7.0 254.59 251.89 109.22 108.06 108.64 DW 258.24 252.07 110.79 108.14 109.46 Acetate pH 5.0 257.00 252.20 110.25 108.19 109.22 pH 5.5 253.54 252.88 108.77 108.49 108.63 pH 6.0 256.80 247.55 110.17 106.20 108.18 pH 6.5 250.91 253.54 107.64 108.77 108.20

[0310] [Table 53]

[0311] Results of purity and dsRNA content studies using elution buffer composition and pH (Gene 1) Elution buffer purity(%) CV (%) dsRNA content (ng / mg) CV (%) First run Second run average First run Second run average citrate pH 5.0 93.96 93.94 93.95 0.19 1.89 1.87 1.88 30.07 pH 5.5 93.86 93.92 93.89 1.03 1.06 1.05 pH 6.0 94.39 94.20 94.30 1.08 1.21 1.14 pH 6.5 94.45 94.25 94.35 1.10 1.09 1.09 pH 7.0 94.42 94.15 94.29 1.03 1.40 1.21 pH 7.5 94.29 94.18 94.24 1.06 1.36 1.21 Tris pH 7.0 94.38 94.33 94.36 1.27 1.74 1.51 DW 94.28 94.17 94.23 2.49 2.72 2.61 Acetate pH 5.0 94.17 94.39 94.28 1.73 2.02 1.88 pH 5.5 94.18 94.41 94.30 2.02 1.61 1.82 pH 6.0 93.84 93.90 93.87 2.07 1.90 1.98 pH 6.5 94.29 94.30 94.30 1.79 2.18 1.99

[0312] [Table 54] Purity results of stability study (gene 1) Elution buffer 0 days (%) Room temperature (4 days) (%) CV (%) 1) Refrigerated for 7 days (%) CV (%) 1) First run Second run average First run Second run average First run Second run average citrate pH 5.0 93.96 93.94 93.95 94.68 94.93 94.81 0.64 94.98 95.03 95.01 0.79 pH 5.5 93.86 93.92 93.89 94.74 95.01 94.88 0.74 95.40 95.21 95.31 1.06 pH 6.0 94.39 94.20 94.30 94.62 94.89 94.76 0.34 95.10 94.72 94.91 0.46 pH 6.5 94.45 94.25 94.35 94.75 95.06 94.91 0.41 95.55 95.00 95.28 0.69 pH 7.0 94.42 94.15 94.29 94.70 95.08 94.89 0.45 95.53 94.85 95.19 0.68 pH 7.5 94.29 94.18 94.24 94.52 94.19 94.36 0.09 94.85 94.75 94.80 0.42 Tris pH 7.0 94.38 94.33 94.36 94.70 95.07 94.89 0.40 95.11 95.02 95.07 0.53 DW 94.28 94.17 94.23 94.62 95.01 94.82 0.44 94.86 94.86 94.86 0.47 Acetate pH 5.0 94.29 94.30 94.30 95.09 95.09 95.09 0.59 95.00 94.95 94.98 0.51 pH 5.5 94.18 94.41 94.30 94.38 94.59 94.49 0.14 95.27 94.94 95.11 0.60 pH 6.0 93.84 93.90 93.87 94.78 95.13 94.96 0.81 95.48 93.97 94.73 0.64 pH 6.5 94.17 94.39 94.28 95.07 94.37 94.72 0.33 94.77 94.76 94.77 0.36

[0313] 1) CV (%) between the 0-day average and the average at room temperature (4 days), and CV (%) between the 0-day average and the average at refrigeration (7 days).

[0314] On the other hand, for gene 2, as shown in Tables 55 and 56, it was confirmed that QA / PA was not different within the treatable range, regardless of the composition and pH of the elution buffer, with the %CV between yields being less than 2% and the %CV between purities being less than 1%. The dsRNA content of the elution buffer was examined, and it was confirmed that the %CV between dsRNA contents was 21.89%, confirming the existence of differences depending on the composition and pH of the elution buffer. Furthermore, the dsRNA contents in citrate buffer (pH 6.0–7.5) and acetate buffer (pH 6.5) were less than 10 ng / mg compared to other buffers.

[0315] In addition, as shown in Table 57, the storage stability of the eluent in all elution buffers was confirmed to be stable for 4 days and 7 days at room temperature, and the maximum purity %CV was 0.66% between days 0 and 4 at room temperature and between days 0 and 7 at room temperature.

[0316] 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 under refrigeration. However, in order to reduce dsRNA content, citrate buffer (pH 6.0 - 7.5) and acetate buffer (pH 6.5) were determined to be suitable as elution buffers.

[0317] [Table 55]

[0318] Yield results from elution buffer composition and pH studies (Gene 2) Elution buffer Elution volume (ug) Yield (%) CV (%) First run Second run First run Second run average citrate pH 5.0 309.90 309.23 109.31 109.08 109.19 1.49 pH 5.5 308.47 309.64 108.81 109.22 109.01 pH 6.0 313.86 313.56 110.71 110.60 110.66 pH 6.5 316.18 315.71 111.53 111.36 111.44 pH 7.0 321.60 309.02 113.44 109.00 111.22 pH 7.5 309.37 298.68 109.13 105.35 107.24 Tris pH 7.0 313.79 327.24 110.69 115.43 113.06 DW 302.12 317.20 106.57 111.89 109.23 Acetate pH 5.0 313.49 N / A 110.58 N / A 110.58 pH 5.5 311.77 302.01 109.97 106.53 108.25 pH 6.0 316.18 309.80 111.53 109.28 110.40 pH 6.5 304.06 309.66 107.25 109.23 108.24

[0319] [Table 56] Results of purity and dsRNA content (gene 2) used to study the composition and pH of the elution buffer. Elution buffer purity(%) CV (%) dsRNA content (ng / mg) CV (%) First run Second run average First run Second run average citrate pH 5.0 91.59 91.44 91.52 0.27 7.23 14.38 10.81 21.89 pH 5.5 91.43 91.51 91.47 9.48 11.89 10.69 pH 6.0 91.68 91.31 91.50 6.99 8.94 7.96 pH 6.5 91.67 91.36 91.52 6.77 8.22 7.50 pH 7.0 91.51 91.37 91.44 6.87 7.39 7.13 pH 7.5 91.21 91.54 91.38 6.87 6.51 6.69 Tris pH 7.0 91.26 90.97 91.12 8.46 9.42 8.94 DW 90.62 90.81 90.72 8.63 15.29 11.96 Acetate pH 5.0 91.37 N / A 91.37 9.28 13.40 11.34 pH 5.5 91.05 91.20 91.13 8.76 13.32 11.04 pH 6.0 91.27 91.21 91.24 8.23 14.66 11.44 pH 6.5 91.04 91.03 91.04 6.63 6.90 6.76

[0320] [Table 57] Purity results of stability study (gene 2) Elution buffer 0 days (%) Room temperature (4 days) (%) CV (%) 1) Refrigerated for 7 days (%) CV (%) 1) First run Second run average First run Second run average First run Second run average citrate pH 5.0 91.59 91.44 91.52 91.11 91.39 91.25 0.21 91.8 91.81 91.81 0.22 pH 5.5 91.43 91.51 91.47 90.76 91.09 90.93 0.42 91.71 91.66 91.69 0.17 pH 6.0 91.68 91.31 91.50 90.72 91.2 90.96 0.41 91.84 91.89 91.87 0.29 pH 6.5 91.67 91.36 91.52 91.01 90.88 90.95 0.44 91.81 91.55 91.68 0.13 pH 7.0 91.51 91.37 91.44 91.03 91.4 91.22 0.17 91.69 91.71 91.70 0.20 pH 7.5 91.21 91.54 91.38 90.95 91.58 91.27 0.09 91.82 91.78 91.80 0.33 Tris pH 7.0 91.26 90.97 91.12 90.7 91.44 91.07 0.03 91.73 91.52 91.63 0.39 DW 90.62 90.81 90.72 90.58 91.44 91.01 0.23 91.51 91.62 91.57 0.66 Acetate pH 5.0 91.04 91.03 91.04 90.82 90.87 90.85 0.15 91.5 91.6 91.55 0.40 pH 5.5 91.05 91.2 91.13 90.83 91.66 91.25 0.09 91.7 91.58 91.64 0.40 pH 6.0 91.27 91.21 91.24 90.65 90.98 90.82 0.33 91.78 91.68 91.73 0.38 pH 6.5 91.37 N / A 91.37 91.58 N / A 91.58 0.16 91.91 N / A 91.91 0.42

[0321] 1) CV (%) between the 0-day average and the average at room temperature (4 days), and CV (%) between the 0-day average and the average at refrigeration (7 days).

[0322] Furthermore, for gene 3, as shown in Tables 58 and 59, it was confirmed that QA / PA was indistinguishable within the manageable range, regardless of the composition and pH of the elution buffer, with %CV between yields within 2% and %CV between purities within 1%. The dsRNA content of the elution buffer was also examined, and it was confirmed that the %CV between dsRNA contents was 17.72%, which is within the method variability level (standard: %CV ≤ 20). Therefore, it was determined that the dsRNA content would not vary with the elution buffer.

[0323] In addition, as shown in Table 60, it was confirmed that, except for the citrate buffer (pH 7.0), the storage stability of the eluent was stable at room temperature for 4 days, 4 days and 7 days, with a purity %CV of less than 2%.

[0324] That is, all buffers meet the acceptance criteria and QA / PA will not differ from the elution buffer, but all buffers except citrate buffer (pH 7.0) are deemed suitable because the room temperature stability of citrate buffer (pH 7.0) is reduced.

[0325] [Table 58] Yield results used for elution buffer composition and pH studies (Gene 3) Elution buffer Elution volume (ug) Yield (%) CV (%) First run Second run First run Second run average citrate pH 5.0 213.15 223.79 93.12 97.77 95.44 1.18 pH 5.5 213.27 226.99 93.17 99.17 96.17 pH 6.0 212.07 223.18 92.65 97.50 95.08 pH 6.5 226.92 219.12 99.14 95.73 97.43 pH 7.0 216.24 223.42 94.47 97.61 96.04 pH 7.5 222.20 223.37 97.07 97.59 97.33 Tris pH 7.0 221.66 222.61 96.84 97.25 97.04 DW 217.39 219.95 94.97 96.09 95.53 Acetate pH 5.0 225.56 223.30 98.54 97.56 98.05 pH 5.5 225.57 220.74 98.55 96.44 97.49 pH 6.0 225.75 225.72 98.62 98.61 98.62 pH 6.5 218.19 220.54 95.32 96.35 95.83

[0326] [Table 59] Results of purity and dsRNA content (gene 3) used for elution buffer composition and pH studies Elution buffer purity(%) CV (%) dsRNA content (ng / mg) CV (%) First run Second run average First run Second run average citrate pH 5.0 80.08 79.87 79.98 0.41 12.65 15.71 14.18 17.72 pH 5.5 80.02 80.04 80.03 10.50 10.19 10.35 pH 6.0 80.11 79.90 80.01 11.02 7.28 9.15 pH 6.5 80.27 80.17 80.22 10.24 11.39 10.82 pH 7.0 79.79 78.25 79.02 8.94 5.26 7.10 pH 7.5 80.16 80.09 80.13 12.16 5.05 8.60 Tris pH 7.0 79.37 79.66 79.52 10.16 11.21 10.68 DW 79.83 79.55 79.69 10.75 10.91 10.83 Acetate pH 5.0 80.00 79.61 79.81 11.03 9.58 10.30 pH 5.5 80.06 79.69 79.88 13.13 10.50 11.81 pH 6.0 79.68 79.66 79.67 9.29 8.14 8.72 pH 6.5 79.94 79.39 79.67 13:30 10.20 11.75

[0327] [Table 60] Purity results used for stability studies (gene 3) Elution buffer 0 days (%) Room temperature (4 days) (%) CV (%) 1) Refrigerated for 7 days (%) CV (%) 1) First run Second run average First run Second run average First run Second run average citrate pH 5.0 80.08 79.87 79.98 78.35 78.72 78.54 1.28 79.69 79.51 79.60 0.33 pH 5.5 80.02 80.04 80.03 77.69 79.48 78.59 1.29 79.54 79.83 79.69 0.31 pH 6.0 80.11 79.9 80.01 77.84 79.28 78.56 1.29 79.91 79.74 79.83 0.16 pH 6.5 80.27 80.17 80.22 78.86 79.26 79.06 1.03 79.58 79.86 79.72 0.44 pH 7.0 79.79 78.25 79.02 73.93 74.88 74.41 4.25 78.29 75.6 76.95 1.88 pH 7.5 80.16 80.09 80.13 78.54 79.33 78.94 1.06 79.71 79.86 79.79 0.30 Tris pH 7.0 79.37 79.66 79.52 78.57 78.76 78.67 0.76 N / A 79.75 77.64 0.21 DW 79.83 79.55 79.69 79.2 78.88 79.04 0.58 79.81 79.82 79.82 0.11 Acetate pH 5.0 79.94 79.39 79.67 79.51 79.02 79.27 0.36 79.84 N / A 76.46 0.16 pH 5.5 80.06 79.69 79.88 79.02 79.09 79.06 0.73 79.87 79.02 79.45 0.38 pH 6.0 79.68 79.66 79.67 77.54 78.86 78.20 1.32 78.75 79.49 79.12 0.49 pH 6.5 80 79.61 79.81 78.09 77.5 77.80 1.80 79.87 79.46 79.67 0.12

[0328] 1) CV (%) between the 0-day average and the average at room temperature (4 days), and CV (%) between the 0-day average and the average at refrigeration (7 days).

[0329] In summary, it has been confirmed that the citrate buffer (pH 5.5 - 7.5) is suitable for gene 1, the citrate buffer (pH 6.0 - 7.5) and the acetate buffer (pH 6.5) are suitable for gene 2, and all buffers except the citrate buffer (pH 7.0) are suitable as elution buffers for gene 3. Therefore, considering all three constructs, the citrate buffer (pH 6.0 - 6.5) and the citrate buffer (pH 7.5) are determined to be suitable for all constructs.

[0330] Although the invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that the description is merely a preferred embodiment and does not limit the scope of the invention. Therefore, the substantial scope of the invention will be defined by the appended claims and their equivalents.

[0331] Industrial Applicability

[0332] The ssRNA purification method according to the present invention has a high mRNA recovery rate and a dsRNA removal rate of 95% or more, which is higher than existing dsRNA removal methods (IP / RP, etc.). Therefore, it can produce mRNA samples with dsRNA content similar to that of mutant T7 RNA polymerase (which can significantly reduce the amount of dsRNA). Furthermore, the amount of dsRNA generated during mRNA synthesis varies depending on the sequence; for some sequences, excessive dsRNA may be generated, making the sequence unsuitable for drug use. However, the ssRNA purification method according to the present invention is very useful because it can minimize or appropriately control the dsRNA content in the mRNA sample, thereby enabling the development of mRNA into drugs regardless of sequence type and maximizing patient safety and drug efficacy. [Simplified Explanation of the Diagram]

[0014] Figure 1 is a schematic diagram illustrating the ssRNA purification process according to the present invention.

[0015] Figure 2 shows the size exclusion chromatography results based on Superdex 200 PG resin according to an embodiment of the present invention.

[0016] Figure 3 shows the size exclusion chromatography results based on Superose 6 PG resin according to an embodiment of the present invention.

[0017] Figure 4 shows the size exclusion chromatography results based on Sepharose 6 FF resin according to an embodiment of the present invention.

[0018] Figure 5 shows the size exclusion chromatography results based on Sephacryl S-300 HR resin according to an embodiment of the present invention.

[0019] Figure 6 shows the comparison results between size exclusion chromatography based on Superose 6 PG resin and size exclusion chromatography based on Sepharose 6 FF resin performed under the same conditions according to an embodiment of the present invention.

[0020] Figure 7 shows the stepwise elution results in C4-HLD resin hydrophobic interaction chromatography according to an embodiment of the present invention.

[0021] Figure 8 shows the gradient elution results of hydrophobic interaction chromatography based on Benzyl Ultra resin according to an embodiment of the present invention.

[0022] Figure 9 shows the gradient elution results of hydrophobic interaction chromatography based on Capto Phenyl (highly substituted) resin according to an embodiment of the present invention.

[0023] Figure 10 is a schematic diagram illustrating an ssRNA purification process according to an embodiment of the present invention.

[0024] Figure 11 shows the results of measuring the level of IFN-σ, a factor related to the innate immune response in mice, with dsRNA concentration via ELISA according to an embodiment of the present invention.

[0025] Figure 12 illustrates the results of measuring the changes in dsRNA concentration of factors related to the innate immune response in mice according to an embodiment 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 by flow cytometry, respectively.

[0026] Figure 13 shows the results of measuring the level of IFN-β, a factor related to the innate immune response in mice, with dsRNA concentration via ELISA according to an embodiment of the present invention.

[0027] Figure 14 illustrates the results of measuring the changes in dsRNA concentration of factors related to the innate immune response in mice according to an embodiment 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.

[0028] Figure 15 illustrates the results of evaluating the dsRNA toxicity in mice as a function of dsRNA concentration according to an embodiment of the present invention. Specifically, (A) and (B) show the results of measuring ALT and AST secretion levels, respectively.

Claims

1. A method for purifying single-stranded RNA (ssRNA), comprising the steps of: (a) performing initial purification of a sample containing ssRNA and double-stranded RNA (dsRNA) as impurities using affinity chromatography; and (b) performing secondary purification of the sample using at least one chromatographic method selected from the group consisting of size exclusion chromatography (SEC), anion exchange chromatography (AEX), and hydrophobic interaction chromatography (HIC).

2. The method as described in claim 1, wherein, The sample was obtained through in vitro transcription (IVT).

3. The method as described in claim 1, wherein, The step (a) of the preliminary purification of the sample using affinity chromatography includes: (ai) mixing the sample containing ssRNA with a sample preparation buffer and loading the mixture onto a column containing a resin that binds complementary to the poly(A) tail; (a-ii) washing the column with a washing buffer; and (a-iii) recovering the bound ssRNA by elution with an elution buffer.

4. The method as described in claim 3, wherein, The sample preparation buffer is a solution containing the following (pH 6.0 to 7.0): 10 to 150 mM sodium phosphate, tris(hydroxymethyl)aminomethane, sodium citrate or sodium acetate; 1 to 20 mM EDTA (ethylenediaminetetraacetic acid); and 100 to 1,000 mM sodium chloride.

5. The method as described in claim 3, wherein, Step (a) further includes an additional loading equilibration buffer step prior to steps (a-ii).

6. The method as described in claim 5, wherein, The equilibration buffer is a solution containing the following (pH 6.0 to 7.0): 10 to 100 mM sodium phosphate, tris(hydroxymethyl)aminomethane (Tris), sodium citrate or sodium acetate; 1 to 10 mM EDTA (ethylenediaminetetraacetic acid); and 100 to 500 mM sodium chloride.

7. The method as described in claim 3, wherein, The washing buffer is a solution containing (pH 6.0 to 7.0) the following: 10 to 100 mM sodium phosphate, Tris, sodium citrate or sodium acetate; and 1 to 10 mM EDTA.

8. The method as described in claim 3, wherein, The elution buffer is a solution containing 0.01 to 10 mM sodium phosphate, Tris, sodium citrate, or sodium acetate (pH 6.0 to 7.0).

9. The method as described in claim 1, wherein, Step (b) of the secondary purification of the sample using size exclusion chromatography includes the following steps: (b-1-i) mixing the pre-purified sample eluted in step (a) with 2X equilibration buffer and loading the mixture into a column containing size exclusion resin; (b-1-ii) eluting the bound RNA with the equilibration buffer; and (b-1-iii) obtaining the final eluted RNA as a fraction containing ssRNA.

10. The method as described in claim 9, wherein, The size exclusion resin separates molecules from 1,500 to 5,000 kDa according to size.

11. The method as described in claim 9, wherein, The equilibration buffer is a solution containing (pH 3.0 to 7.0) the following: 1 to 100 mM sodium citrate, sodium phosphate, Tris or sodium acetate; and 0.01 to 1,000 mM sodium chloride.

12. The method as described in claim 1, wherein, Step (b) of the secondary purification of the sample using the anion exchange chromatography comprises the following steps: (b-2-i) mixing the preliminarily purified sample eluted in step (a) with a sample preparation buffer and loading the mixture onto a column containing the anion exchange resin; and (b-2-ii) obtaining the eluted flow-through (FT) fraction containing ssRNA.

13. The method as described in claim 12, wherein, The sample preparation buffer is a solution containing the following (pH 7.0 to 8.0): 10 to 50 mM Tris, sodium phosphate, sodium citrate or sodium acetate; 1 to 10 mM EDTA; and 500 to 1,000 mM sodium chloride.

14. The method as described in claim 1, wherein, Step (b) of the secondary purification of the sample using the hydrophobic interaction chromatography includes the following steps: (b-3-i) mixing the preliminarily 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 with the sample preparation buffer; and (b-3-iii) recovering the bound ssRNA by elution with an elution buffer.

15. The method as described in claim 14, wherein, The sample preparation buffer is a solution containing the following (pH 6.5 to 7.5): 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.

16. The method as described in claim 14, wherein, Step (b) further includes an additional sample preparation buffer loading step prior to step (b-3-ii).

17. The method as described in claim 14, wherein, The elution buffer is a solution containing the following (pH 6.5 to 7.5): 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.

18. The method as described in claim 1, further comprising an ultrafiltration step before, after, or after step (b).

19. The method as described in claim 1, wherein, The ssRNA obtained through the secondary purification has a dsRNA removal rate of 80% or more.

Images