Method for purifying lipid nanoparticles

Abstract

The present invention relates to a method for purifying nucleic acid-encapsulating lipid nanoparticles (LNPs), comprising the steps of: providing a solution containing the LNPs to a convective chromatographic medium in the presence of at least one cosmotropic agent; and eluting the LNPs from the chromatographic medium. The present invention further relates to the use of various convective chromatographic mediums for the purification of nucleic acid-encapsulating lipid nanoparticles (LNPs).

Description

Technical Field

[0001] The present invention relates to a method for purifying lipid nanoparticles (LNP) encapsulating nucleic acids, comprising a step of supplying a solution containing the LNP to a chromatographic medium having convective properties in the presence of at least one cosmotropic agent, and a step of eluting the LNP from the chromatographic medium. The present invention further relates to the respective use of a chromatographic medium having convective properties for the purification of lipid nanoparticles (LNP) encapsulating nucleic acids.

Background Art

[0002] LNP encapsulating nucleic acids have emerged as promising prophylactic and / or therapeutic agents, for example, as vaccines carrying messenger RNA (mRNA), self-amplifying RNA (saRNA), or circular RNA (circRNA). LNP typically consists of ionizable cationic lipids, cholesterol, helper lipids, and PEGylated lipids, and typically has a diameter in the range of 30 nm to 300 nm (for example, 80 nm). However, LNP is usually very sensitive to shear.

[0003] For example, the production of encapsulated mRNA for use as a vaccine consists of multiple unit operations including microbial plasmid production, plasmid isolation, linearization, and an in vitro transcription reaction to generate mRNA. The mRNA is purified by precipitation, chromatography, or tangential flow filtration (TFF) and formulated in a low-conductivity buffer and weak acidic conditions. Then, the mRNA is encapsulated into lipid nanoparticles, which are spherical mixtures of mRNA and ionizable lipids, to form particles having a diameter of approximately 100 nm. The formulation of mRNA into LNP is achieved by high-pressure in-line mixing of lipids dissolved in one or more organic solvents such as ethanol, methanol, acetone, isopropanol, ethyl acetate, etc. in a microfluidic chamber.

[0004] ]The LNPs thus produced are initially formulated in a high proportion of organic solvent (typically in the range of 10% (vol / vol) to 50% (vol / vol)), which negatively affects the stability of the nanoparticles and can alter their size. Therefore, the organic solvent must be removed from the formulation by dilution or buffer exchange. This is typically achieved by TFF with a molecular weight cutoff of 30 kDa to 300 kDa. However, this approach generates large shear forces and turbulence, degrading the LNPs and resulting in a reduced recovery rate of the buffer-exchanged LNPs. Furthermore, because the cutoff size is smaller than the typical size of mRNA, unencapsulated mRNA is also purified and enriched along with the LNPs. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, the fundamental technical problem underlying the present invention is to provide a method for purifying LNPs that encapsulate nucleic acids, with improved purity and yield. [Means for solving the problem]

[0006] The solutions to the above technical problems are achieved by embodiments characterized by the claims.

[0007] In particular, in the first embodiment, the present invention is a method for purifying lipid nanoparticles (LNPs) that encapsulate nucleic acids, (a) A step of subjecting the solution containing the above LNP to a chromatographic medium having convection properties in the presence of at least one cosmotropic agent, (b) Washing the chromatography medium with a solution containing at least one cosmotropic agent, (c) A step of eluting LNP from the above chromatography medium, This includes methods.

[0008] As outlined above, LNPs are typically composed of ionizable cationic lipids, cholesterol, helper lipids, and PEGylated lipids, and are typically nanoparticles with a diameter in the range of 30 nm to 300 nm. However, the LNPs that can be purified by the method of the present invention are not particularly limited and include all LNPs that may be of interest for nucleic acid delivery / administration.

[0009] Similarly, the nucleic acids encapsulated in the above LNPs are not particularly limited and encompass any nucleic acids that may be of interest in preventive and / or therapeutic contexts. Each nucleic acid encompasses single-stranded or double-stranded RNA and DNA molecules of any length, including, for example, messenger RNA (mRNA), self-amplified RNA (saRNA), trans-amplified RNA (taRNA), self-replicating RNA (srRNA), circular RNA (circRNA), guide RNA (gRNA), small interfering RNA (siRNA), and mixtures thereof, as a single species or a combination of two or more species, with gRNA, mRNA, saRNA, and / or circRNA being particularly preferred. The nucleic acids encapsulated in the above LNPs may also include any combination of two or more encapsulated nucleic acids, such as gRNA and mRNA, or DNA and mRNA.

[0010] In step (a) of the method of the present invention, a solution containing LNPs is subjected to a chromatographic medium having convection properties in the presence of at least one cosmotropic agent.

[0011] As used herein, the term “convective chromatographic medium” refers to a chromatographic medium suitable for convective transport, i.e., transport of a solution containing LNPs by convection, for example by gravity or a pump, and not dominated by diffusive transport. Preferably, the convective chromatographic medium has low hydrophobicity and, more preferably, hydrophobicity that allows LNPs to bind to the chromatographic medium in the presence of at least one of the cosmotropic agents, but prevents impurities, such as potentially present nucleic acids in the solution containing LNPs, from binding to the chromatographic medium. Suitable materials for the chromatographic medium are not particularly limited and are known in the art. Such materials include synthetic or natural organic polymers. Preferably, the chromatographic medium is selected from the group consisting of unmodified or modified styrene-divinylbenzene-based materials, unmodified or modified polymethacrylate-based materials, unmodified or modified cellulose-based materials, and unmodified or modified agarose-based materials. Chromatographic supports can be functionalized with hydroxyl, C4, C6, C8, C12, C18, phenyl, or other ligands that exhibit hydrophobic behavior in the presence of cosmotropic salts. Each chromatographic medium may be in the form of a convective chromatographic support such as a monolith, a membrane, nanofibers having a pore size of at least 0.3 μm to a maximum of 6 μm, preferably 1 μm to 2 μm, and porous particles having a pore size of at least 0.3 μm to a maximum of 10 μm, preferably about 1 μm and / or channel size. In a preferred embodiment, the chromatographic medium is a monolithic chromatographic medium functionalized with a hydroxyl ligand. Thus, the chromatographic medium may be a monolithic chromatographic medium, a membrane, nanofibers, the porous particles defined above, or any other chromatographic apparatus exhibiting convective transport of solutes.

[0012] The cosmotropic agent used in step (a) of the method of the present invention is preferably selected from the group consisting of tetramethylammonium, ammonium, potassium, sodium, cesium, lithium, calcium, magnesium, guanidine, citrate, perchlorate, nitrate, thiocyanate, fluoride, chloride, sulfate, carbonate, phosphate including pyrophosphate, carboxylate, and cosmotropic salts of combinations thereof. More specifically, the cosmotropic salt may be tetrasodium pyrophosphate (Na4P2O7), monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), tripotassium phosphate (K3PO4), ammonium sulfate ((NH4)2SO4), sodium chloride (NaCl), monosodium citrate (NaC6H7O7), disodium citrate (Na2C6H6O7), trisodium citrate (Na3C6H5O7), or combinations thereof.

[0013] The present invention uses a chromatographic medium having convective properties in the presence of at least one cosmotropic agent, i.e., under specific hydrophobic interaction chromatography (HIC) conditions, which enable LNPs to selectively bind to the medium. Such HIC conditions include the presence of a cosmotropic agent at a concentration of at least 0.01 M, preferably at least 0.05 M, more preferably 0.1 M to 1 M, more preferably 0.3 M to 0.5 M, for example, about 0.4 M. Furthermore, such HIC may further include a conductivity of at least 5 mS / cm and / or a pH in the range of pH 4 to pH 10, preferably pH 7 to pH 9, more preferably pH 7.4 to pH 8.0.

[0014] In certain embodiments, step (a) of the method of the present invention is preferably carried out at ambient temperature, for example, at a temperature of about 20°C to about 25°C.

[0015] In step (b) of the method of the present invention, the chromatography medium is washed with a solution containing at least one cosmotropic agent.

[0016] The cosmotropic agent used in step (b) of the method of the present invention is preferably selected from the group consisting of tetramethylammonium, ammonium, potassium, sodium, cesium, lithium, calcium, magnesium, guanidine, citrate, perchlorate, nitrate, thiocyanate, fluoride, chloride, sulfate, carbonate, phosphate including pyrophosphate, carboxylate, and cosmotropic salts of combinations thereof. More specifically, the cosmotropic salt may be tetrasodium pyrophosphate (Na4P2O7), monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), tripotassium phosphate (K3PO4), ammonium sulfate ((NH4)2SO4), sodium chloride (NaCl), monosodium citrate (NaC6H7O7), disodium citrate (Na2C6H6O7), trisodium citrate (Na3C6H5O7), or combinations thereof. Preferably, the cosmotropic agent used in step (b) of the method of the present invention is the same as the cosmotropic agent used in step (a) of the method of the present invention.

[0017] Furthermore, the solution containing at least one cosmotropic agent used in step (b) of the method of the present invention preferably contains a cosmotropic agent at a concentration of at least 0.01 M, preferably at least 0.05 M, more preferably 0.1 M to 1 M, and more preferably 0.2 M to 0.3 M. Furthermore, the above solution may have a conductivity of at least 5 mS / cm and / or a pH in the range of pH 4 to pH 10, preferably pH 7 to pH 9, and more preferably pH 7.4 to pH 8.0. Preferably, in the above solution, the concentration and / or conductivity and / or pH of the cosmotropic agent is the same as the concentration and / or conductivity and / or pH of the cosmotropic agent used in step (a) of the method of the present invention.

[0018] In certain embodiments, step (b) of the method of the present invention is preferably carried out at ambient temperature, for example, at a temperature of about 20°C to about 25°C. In connection with this, the above temperature is preferably the same as the temperature in step (a) of the method of the present invention.

[0019] In step (c) of the method of the present invention, LNPs are eluted from the chromatographic medium, preferably by reducing the concentration of the cosmotropic agent. Preferably, the eluent used in this step is water, a solution without a cosmotropic agent, or a solution containing at least one cosmotropic agent at a low concentration used in step (a), or a buffer with a pH in the range of pH 4 to pH 9, such as Tris buffer, the buffer either does not contain a cosmotropic agent or contains at least one cosmotropic agent at a low concentration. If present, such elution may involve the presence of a cosmotropic agent at a concentration of at least 0.0001 M, preferably 0.0005 M to 0.002 M, more preferably about 0.001 M.

[0020] In certain embodiments, the solution containing LNPs used in the method of the present invention contains an organic solvent obtained from a preceding LNP generation process, such as ethanol. Therefore, the method of the present invention may further include a step of diluting the solution containing LNPs with a solution containing at least one cosmotropic agent before step (a). As an example, the solution containing LNPs may typically have an organic solvent concentration of about 10% (vol / vol) to about 80% (vol / vol), preferably about 10% (vol / vol) to about 50% (vol / vol), before dilution, and after dilution, the diluted solution may have an organic solvent concentration of 10% (vol / vol) or less.

[0021] In a second aspect, the present invention relates to the use of a chromatographic medium having convection properties for the purification of lipid nanoparticles (LNPs) encapsulating nucleic acids, wherein the chromatographic medium is used in the presence of at least one cosmotropic agent.

[0022] In this embodiment, all relevant limitations defined for the first embodiment of the present invention also apply to the second embodiment of the present invention. In particular, the convective chromatographic media, LNPs, nucleic acids, and cosmotropic agents are as defined above.

[0023] As used herein, the term "comprising" explicitly includes the terms "consisting essentially of" and "consisting of", that is, in this specification, these terms can all be used interchangeably without distinction from each other.

[0024] Furthermore, as used herein, the term "about" preferably represents a modifier of a specified value of ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.5%. Thus, by way of example, the term "about 100" can include ranges from 90 to 110, 92 to 108, 94 to 106, 95 to 105, 96 to 104, 97 to 103, 98 to 102, 99 to 101, or 99.5 to 100.5.

[0025] The present invention provides a purification method for enhancing the yield and purity of LNPs encapsulating nucleic acids such as mRNA.

[0026] Specifically, the present invention provides an approach for concentrating, buffer-exchanging, and purifying LNPs in a single step. Convective chromatography media such as monoliths, membranes, and nanofibers are composed of interconnected flow-through channels functionalized with ligands. Porous particles having large pores (pore size or channel size greater than 0.3 μm) are also regarded as convective chromatography media. Due to the laminar flow characteristics of the mobile phase flowing through the interconnected channels or large pores, the analyte is exposed to a relatively low shear force compared to TFF. By selecting an appropriate ligand, selectivity for the target analyte can be achieved even under low shear conditions.

[0027] The present invention achieves concentration, purification, and buffer exchange of post-encapsulation LNPs by combining a low shear force environment with a ligand chemistry that binds to LNPs but not to mRNA or other free nucleic acid strands.

[0028] The present invention achieves buffer exchange by binding LNPs in the presence of a cosmotropic salt, such as potassium phosphate, and eluting them with a low concentration of the cosmotropic salt in the presence of buffer or water, using a chromatographic medium with convective properties and low hydrophobicity under hydrophobic interaction chromatography (HIC) conditions. LNPs formulated in a solution with a high concentration of organic solvent (i.e., the product of the mounting step, e.g., having an organic solvent concentration of about 10% (vol / vol) to about 80% (vol / vol)) are first diluted with a mobile phase containing the cosmotropic salt and introduced into a chromatograph in the same mobile phase at ambient temperature. Due to the convective properties of the chromatographic medium, the binding capacity to LNPs is independent of the flow rate, and as a result, the flow rate during loading can be high and the loading time can be short, and therefore, a high initial dilution of LNPs required to dilute the organic solvent in the mixture is not a limitation. In another embodiment, LNPs (i.e., the product of the encapsulation step) formulated in a solution with a high concentration of organic solvent are first diluted with a low conductivity matrix (e.g., PBS or Tris buffer) and then incorporated into the loading buffer by in-line dilution immediately before loading onto the chromatography medium. As in the previous embodiment, the degree of dilution of the LNPs can be increased without affecting the purification time or efficiency. The hydrophobic matrix (e.g., hydroxyl) of the chromatography medium retains the hydrophobic LNPs during loading. However, since the hydrophobicity of contaminating mRNA or other non-encapsulated nucleic acids is lower than that of LNPs, the contaminating mRNA or other non-encapsulated nucleic acids are not retained by the chromatography unit. The LNPs are then eluted into the low conductivity buffer or water in a gradient or step (Figure 1). Due to the convection characteristics of the chromatography medium, the elution of LNPs is not governed by diffusion limitations that lead to broadening of the chromatographic peaks, and a small amount of eluent (e.g., 3-column volume to 5-column volume) is required to achieve elution from the chromatography medium, resulting in a high concentration of LNPs in the eluted fraction and thus enrichment of the LNPs.

[0029] Applying this characteristic of a chromatographic medium with convection properties offers various advantages to the economics and efficiency of downstream purification of LNPs. First, by minimizing the exposure of LNPs to destructive shear forces, the yield of LNP production is increased compared to methods used in the art. Second, chemical identification of the target analyte and impurities (e.g., mRNA) increases the purification factor compared to methods used in the art, which is not currently achieved with methods used in the art (e.g., TFFs that cannot distinguish LNPs from nucleic acid strands based on molecular weight). This increases the purity of the final product, thereby improving clinical efficacy and reducing immunogenicity from residual mRNA. Finally, the method of the present invention is scalable to industrial (multigram) production scale.

[0030] The value of this technology is very high. mRNA or other nucleic acid strands encapsulated in LNPs are increasingly being used as stand-alone vaccines for multiple indications. The demand for LNP production is high, and as a result, there is a growing need to improve the yield of the production / purification process. Current approaches used in the art are not optimal because they lead to particle destruction and do not remove significant impurities. By applying the method described herein, the yield of the LNP process and the purity of the product can be increased, thereby improving the overall recovery rate of the process. [Brief explanation of the drawing]

[0031] [Figure 1]This is a schematic diagram of the purification of lipid nanoparticles (LNPs) that encapsulate nucleic acids, along with any contaminating free nucleic acid species. 1) A sample containing LNPs, free (unencapsulated) RNA, residual organic solvent, and lipids is loaded onto a hydrophobic chromatography medium in the presence of at least one cosmotropic agent. 2) The washing step elutes the free RNA, residual organic solvent, and lipids from the column while the LNPs remain bound. 3) When the eluent is switched to water (or a solution containing a low concentration of at least one cosmotropic agent, or a buffer with a pH in the range of pH 4 to pH 9), the LNPs are released from the chromatography medium. [Figure 2] A: Preparative chromatogram of LNP purification using a CIMmultus OH monolith (bed volume 1 mL, channel diameter 2 mm) with potassium phosphate as the cosmotropic salt. Trace A: UV absorbance at 280 nm, Trace B: UV absorbance at 260 nm, Trace C: Conductivity [mS / cm], Trace D: Multi-angle light scattering (MALS). Load fraction, wash fraction, and elution fraction are shown. B: Magnified view of the loading stage of LNP purification using a CIMmultus OH monolith with potassium phosphate as the cosmotropic salt. Trace A: UV absorbance at 280 nm, Trace B: UV absorbance at 260 nm, Trace C: Conductivity [mS / cm], Trace D: Multi-angle light scattering (MALS). An increase in signals is observed at 260 nm and 280 nm, but MALS does not increase, suggesting a breakthrough of free (unencapsulated) mRNA. [Figure 3] Analytical reverse-phase (CIMac SDVB) chromatograms of i) mRNA standard, ii) LNP standard, iii) flow-through fraction (FT) of LNP preparation by CIMmultus OH purification, and iv) eluted fraction (E) by CIMmultus OH purification. The flow-through fraction shows the presence of species with a retention time of approximately 2.5 minutes, which is consistent with the mRNA elution profile. The eluted fraction shows the presence of species with a retention time of approximately 6.9 minutes, which is consistent with the LNP elution profile. [Figure 4]Preparative chromatograms of LNP purification using a CIMmultus OH monolith (bed volume 1 mL, channel diameter 2 μm) with sodium citrate as the cosmotropic salt, as described in Example 2. Trace A: UV absorbance at 260 nm, Trace B: Multi-angle light scattering (MALS). Load fraction, wash fraction, and elution fraction are shown. [Figure 5] Analytical reverse-phase chromatograms of the load fraction, flow-through fraction (FT), and elution fraction (E) from CIMmultus OH purification of the LNP preparation described in Example 2. The flow-through fraction shows the presence of species with a retention time of approximately 2.8 minutes, which is consistent with the mRNA elution profile. The elution fraction shows the presence of species with a retention time of approximately 7.1 minutes, which is consistent with the LNP elution profile. [Figure 6] Preparative chromatograms of LNP purification using a CIMmultus OH monolith (bed volume 1 mL, channel diameter 2 μm) with LNPs diluted in-line with sodium citrate as a cosmotropic salt, as described in Example 3. Trace A: UV absorbance at 260 nm, Trace B: Multi-angle light scattering (MALS). Load fraction, wash fraction, and elution fraction are shown. [Figure 7] Analytical reverse-phase chromatograms of the loaded fraction, flow-through fraction (FT), and eluted fraction (E) from CIMmultus OH purification of LNPs loaded using the in-line dilution procedure described in Example 3. The flow-through fraction shows the presence of species with a retention time of approximately 2.8 minutes, which is consistent with the mRNA elution profile. The eluted fraction shows the presence of species with a retention time of approximately 7.1 minutes, which is consistent with the LNP elution profile. [Figure 8] Nanoparticle tracking analysis (NTA) was performed to demonstrate the particle size distribution of a) the loaded fraction and b) the eluted fraction of LNP purified using a CIMmultus OH monolith, with LNPs diluted in-line with sodium citrate as a cosmotropic salt, as described in Example 3. [Figure 9]Preparative chromatogram of LNP purification using a CIMac® OH monolith (bed volume 0.1 mL, channel diameter 6 mm) with sodium citrate as the cosmotropic salt. Dotted traces: UV absorbance at 260 nm, dashed traces: UV absorbance at 280 nm, solid traces: multi-angle light scattering (MALS). Load fraction, elution fraction, and settling wash fraction are shown. The chromatogram was acquired using a PATfix® LC system, and data acquisition and analysis were performed using PATfix® software. Preparation and purification are described in Example 4. [Figure 10] This is an analytical chromatogram of an LNP sample from 2D chromatography, showing the ion-pair reversed-phase chromatography portion. Dotted trace: blank UV absorbance at 260 nm, dashed trace: UV absorbance of the crude LNP formulation at 260 nm, solid trace: UV absorbance of the eluted LNP sample at 260 nm. The first peak represents bare mRNA eluted as the acetonitrile concentration increases. The second peak represents encapsulated mRNA eluted as the acetonitrile concentration increases. The chromatogram was acquired using a PATfix® LC system, and data acquisition and analysis were performed using PATfix® software. Preparation and purification are described in Example 4. [Figure 11] A: Shows the sample size and concentration analysis from Myriade Videodrop measurement of the crude LNP formulation described in Example 4. B: Shows the sample size and concentration analysis from Myriade Videodrop measurement of the eluted LNP sample described in Example 4. This shows that the size remains almost the same, while the concentration has increased (recalculation is required due to dilution, which is described in Example 4). [Figure 12]Cryo-TEM analysis of LNPs: A: Main eluted sample from Example 4, B: Control particles with only buffer replacement after compounding, C: Flow-through from preparative chromatography. This shows no significant change in particles on the OH column, but in the flow-through only particles with low electron density are observed, indicating empty particles. The analysis was performed using a JEM-1230 TEM (JEOL) microscope equipped with a Gatan Ultrascan 4000 SP CCD detector. Samples were prepared by dispensing onto an L-polylysine grid before quenching in liquid ethane. [Figure 13] Preparative chromatogram of LNP purification using a CIMac® C4 monolith (bed volume 0.1 mL, channel diameter 2 mm) with sodium citrate as the cosmotropic salt. Traces are indicated within the legend box in the figure. The broad peaks in the MALS trace are elution peaks observed when conductivity is reduced. The sharp peaks after the elution peaks are settling wash peaks. The peaks at high conductivity at the start of the chromatogram indicate flow-through fractionation, and the UV trace clearly shows free mRNA (UV260 / 280 ratio = 2). Chromatograms were acquired using a PATfix® LC system, and data acquisition and analysis were performed using PATfix® software. Preparation and purification are described in Example 5. [Figure 14] Preparative chromatogram of LNP purification using Poros 50 OH resin with sodium citrate as the cosmotropic salt via the in-line dilution procedure described in Example 3. Trace A: UV absorbance at 280 nm, Trace B: UV absorbance at 260 nm, Trace C: conductivity [mS / cm], Trace D: Multi-angle light scattering (MALS). Load fraction, wash fraction, and elution fraction are shown. [Figure 15] Nanoparticle tracking analysis (NTA) was performed to demonstrate the particle size distribution of a) the loaded fraction and b) the eluted fraction of LNP purified using Poros OH resin, with LNPs diluted in-line with sodium citrate as the cosmotropic salt, as described in Example 6. [Modes for carrying out the invention]

[0032] The present invention will be further illustrated by the following embodiments, but will not be limited thereto. [Examples]

[0033] Example 1: Purification of LNPs using CIMmultus OH in potassium phosphate Following the manufacturer's protocol (N / P ratio 4, aqueous solution to lipid solution flow rate ratio 3:1, total flow rate 12 mL / min, final sample volume 0.5 mL), approximately 100 μg of mRNA encoding eGFP (995 nucleotides) (starting concentration 0.2 mg / mL) was encapsulated in lipid nanoparticles using NanoAssemblr™ Ignite™ with Genvoy ILM lipid mix. The LNP sample was directly diluted in 1 M potassium phosphate (total volume 12 mL) and loaded onto CIMmultus OH (1 mL, pore size 2 μm). During sample loading, the UV 260 / 280 nm signal gradually increased, but no increase in the MALS signal was observed (Figure 2B). After sample loading was complete, the column was washed with mobile phase A (1 M potassium phosphate) until the UV signal returned to baseline. Elution to 100% ddH2O was performed in a single step. Significant increases in both the UV 260 / 280 nm and MALS signals were observed.

[0034] Analysis of the samples corresponding to the flow-through fraction and elution fraction by reverse-phase HPLC (Figure 3) showed that RNA was present in the flow-through fraction but LNPs were not, while LNPs were present in the elution fraction but RNA was not.

[0035] Example 2: Purification of LNPs with CIMmultus OH in sodium citrate Following the procedure described above, 0.2 mg of mRNA encoding eGFP (995 nucleotides) was mounted. The LNP product (1.6 mL) was immediately diluted 10-fold with PBS to reduce the ethanol concentration to less than 3%. 15 mL of this sample was diluted with mobile phase A (15 mM Tris, 250 mM sodium citrate, pH 7.51) to match the conductivity and pH of the MPA, and prepared for loading. After loading the sample, the column was washed with 10 mL of MPA. Elution was performed stepwise from 100% MPA to 100% MPB (15 mM Tris, pH 7.52), and 100% MPB was maintained over 10 mL (10 column volumes). The column strip was rinsed with deionized water (dH2O; 10 mL), and the column was sanitized with 1 M NaOH (5 column volumes). Chromatographic separation is shown in Figure 4. A slight increase in the MALS signal at 15 mL–20 mL indicates LNP breakthrough (excess binding capacity). A significant increase in UV260 and MALS signals was observed during elution in MPB (30 mL). Reverse-phase chromatography analysis showed that free (unencapsulated) mRNA was present in the flow-through but not in the eluted fraction, and that LNPs were present in the eluted fraction (E) but not in the flow-through (Figure 5). Ribogreen analysis of the fraction (comparison of Ribogreen fluorescence signals of RNA before and after washing with a washing agent to release encapsulated RNA) showed that the LNP elution recovery rate was at least 60%. Nanoparticle tracking analysis showed the presence of particles with diameters of 140 nm–185 nm, confirming the presence of LNPs during CIMmultus OH elution.

[0036] Example 3: Purification of LNPs with CIMmultus OH in sodium citrate by in-line dilution Following the manufacturer's protocol (N / P ratio 6, aqueous solution to lipid solution flow ratio 3:1, total flow rate 12 mL / min, final sample volume 1.6 mL), approximately 0.2 mg of 4000 nucleotide mRNA (starting concentration 0.1 mg / mL) was encapsulated in lipid nanoparticles using NanoAssemblr® Ignite® with Genvoy ILM lipid mix. The LNP product (1.6 mL) was immediately diluted 10-fold with PBS to reduce the ethanol concentration to less than 3%. 15 mL of this sample was loaded by in-line dilution with dilution buffer (30 mM Tris, 500 mM sodium citrate, pH 7.5) to match the conductivity and pH of MPA (15 mM Tris, 250 mM sodium citrate, pH 7.51). After loading the sample, the column was washed with 6 mL of MPA. Elution was performed stepwise from 100% MPA to 100% MPB (dH2O), and 100% MPB was retained for 12 mL (12 column volumes). The columns were sanitized with 1 M NaOH (5 column volumes). Chromatographic separation is shown in Figure 6. A significant increase in UV260 and MALS signals was observed in the 100% MPB (elution) step, suggesting LNP elution. Reverse-phase chromatography analysis showed a low signal corresponding to free (unencapsulated) mRNA in the flow-through fraction, but not in the eluted fraction, and indicated the presence of LNPs in the eluted fraction (E) but not in the flow-through fraction (FT) (Figure 7). Ribogreen analysis of the fractions (comparison of Ribogreen fluorescence signals of RNA before and after washing with a washing agent to release encapsulated RNA) showed an LNP elution recovery rate of 95%. Nanoparticle tracking analysis of the load revealed the presence of a broad particle size distribution with a mode value of 201 nm, and the concentration during loading was 4.4 × 10⁻⁶. 9 The particle count was 4.5 × 10¹⁶ particles / mL (Figure 8a). The elution fraction (E) showed the presence of particles with a mode value diameter of 142 nm (Figure 8b), and the particle concentration was 4.5 × 10¹⁶. 10 The particle concentration was 10 times higher than that of the load, confirming the presence of LNPs.

[0037] Example 4: Purification of LNPs with CIMac OH in sodium citrate by in-line dilution 4000 nucleotide mRNA (128 μg, 170 μg / mL in 100 mM NaOAc, pH 4.0) was encapsulated in lipid nanoparticles using NanoAssemblr® Ignite®. The aqueous stream was mixed with a lipid solution stream (molar ratio: ALC-0315:DSPC:PEG-2KDMG:cholesterol = 9.1:1.8:1.0:8.9, 12.5 mM in EtOH solution) at a flow rate ratio of 3:1 and a total flow rate of 2 mL / min. The LNP product (0.8 mL) was immediately diluted 10-fold with PBS to reduce the ethanol concentration to less than 3%. 5 mL of this sample was diluted in-line with dilution buffer (20 mM Tris, 250 mM sodium citrate, pH 7.4) to match the conductivity and pH of MPA (15 mM Tris, 200 mM sodium citrate, pH 7.4) and loaded at a flow rate of 10 CV / min. After loading the sample, the column was washed with 1 mL of MPA. Elution was performed stepwise from 100% MPA to 100% MPB (15 mM Tris, pH 7.4), with 100% MPB held for 5 minutes (50 column volume). The column was sanitized with 1 M NaOH (20 column volume). The chromatographic separation is shown in Figure 9. A significant increase in UV260 and MALS signals was observed in the 100% MPB (elution) step, suggesting LNP elution. Ion-pair reversed-phase chromatography analysis showed a significantly higher proportion of encapsulated mRNA in the eluted fraction compared to the raw sample (Figure 10). Videodrop size measurement analysis of the load revealed the presence of a particle size distribution with a mode value of 115 nm, and the concentration in the load was 1.3 × 10⁻⁶. 10 The particle count was 3.3 × 10¹⁶ particles / mL (Figure 11a). The elution fraction (E) showed the presence of particles with a mode value diameter of 115 nm (Figure 11b), and the particle concentration was 3.3 × 10¹⁶. 10The particle / mL concentration confirmed the presence of LNPs and indicated particle concentration. Analysis of the stationary wash fraction showed the presence of very large particles (fused and aggregated LNPs), but the flow-through fraction had a very low particle concentration (Figure 12c), and most of them were low electron density (empty) particles. Cryo-TEM analysis confirmed that the eluted particles were mostly spherical (Figure 12a), had a high electron density on average, and were morphologically equivalent to control particles (buffer exchange only after compounding, Figure 12b).

[0038] Example 5: Purification of LNPs using CIMac modified with C4 ligand in sodium citrate LNPs were prepared from a 4000-nucleotide mRNA and lipid mixture (molar ratio: SM-102:DSPC:PEG-2KDMG:cholesterol = 50:10:1.5:38.5). The sample was prepared and loaded onto a column with buffer A (100 mM, 15 mM Tris, pH 8.0). Elution was performed with a 30 CV gradient from 100% A to 100% buffer B (15 mM Tris, pH 8.0), and then maintained at 100% B for another 20 CV. The column was sanitized with 1 M NaOH (20 CV). The chromatographic separation is shown in Figure 13. Flow-through peaks are observed in the UV spectrum corresponding to free mRNA from the UV 260 / 280 nm ratio. MALS peaks are eluted during the elution gradient, and some are eluted at the sanitization stage. These correspond to LNPs.

[0039] Example 6: Purification of LNPs with Poros OH in sodium citrate by in-line dilution Following the manufacturer's protocol (N / P ratio 4, aqueous solution to lipid solution flow rate ratio 3:1, total flow rate 12 mL / min, final sample volume 3.8 mL), approximately 0.5 mg of 4000 nucleotide mRNA (starting concentration 0.2 mg / mL) was encapsulated in lipid nanoparticles using NanoAssemblr® Ignite® with Genvoy ILM lipid mix. The LNP product (3.8 mL) was immediately diluted 20-fold with PBS to reduce the ethanol concentration to less than 3%. 70.4 mL of this sample was loaded onto Poros 50 OH resin by in-line dilution using dilution buffer MPA (30 mM Tris, 1.5 M sodium citrate, pH 7.5), and 4.8 mL of the wet sedimentation volume was packed into an XK16 / 20 column. After loading the sample, the column was washed with 60 mL of 50% MPA and 50% MPB (1 × PBS). Elution was performed stepwise down to 100% MPC (dH2O), and the 100% MPC was retained for 25 mL. The column was sanitized with 1 M NaOH and 2 M NaCl. Chromatographic separation is shown in Figure 14. A significant increase in UV260 and MALS signals was observed during the 100% MPC (elution) step, suggesting LNP elution. Ribogreen analysis of the fraction (comparison of Ribogreen fluorescence signals of RNA before and after washing with a washing agent to release encapsulated RNA) showed an LNP elution recovery rate of 78%. Nanoparticle tracking analysis of the load showed the presence of a broad particle size distribution with a mode value of 91 nm, and the concentration during loading was 9.08 × 10⁶. 10 The particle count was 1.54 × 10¹⁶ particles / mL (Figure 15a). The elution fraction (E) showed the presence of particles with a mode value diameter of 118 nm (Figure 15b), and the particle concentration was 1.54 × 10¹⁶. 11 The particle / mL value was confirmed, and the presence of LNPs was verified.

Claims

1. A method for purifying lipid nanoparticles (LNPs) that encapsulate nucleic acids, (a) A step of subjecting the solution containing the LNP to a chromatographic medium having convection properties in the presence of at least one cosmotropic agent, (b) Washing the chromatography medium with a solution containing at least one cosmotropic agent, (c) A step of eluting LNP from the chromatography medium, Methods that include...

2. The method according to claim 1, wherein the chromatography medium exhibits hydrophobicity in the presence of the at least one cosmotropic agent, and the at least one cosmotropic agent enables the LNP to bind to the chromatography medium, but prevents the contamination nucleic acid from binding to the chromatography medium.

3. The method according to claim 1 or 2, wherein the chromatography medium is a synthetic or natural organic polymer.

4. The method according to any one of claims 1 to 3, wherein the chromatography medium is selected from the group consisting of unmodified or modified styrene-divinylbenzene-based materials, unmodified or modified polymethacrylate-based materials, unmodified or modified cellulose-based materials, and unmodified or modified agarose-based materials.

5. The method according to any one of claims 1 to 4, wherein the chromatography medium is functionalized with hydroxyl, C4, C6, C8, C12, C18, or phenyl ligand.

6. The method according to any one of claims 1 to 5, wherein the chromatography medium is a monolithic chromatography medium, a membrane, nanofibers, or porous particles.

7. The method according to any one of claims 1 to 6, wherein the at least one cosmotropic agent is present in the solution at a concentration of at least 0.01 M.

8. The method according to any one of claims 1 to 7, wherein the cosmotropic agent is selected from the group consisting of tetramethylammonium, ammonium, potassium, sodium, cesium, lithium, calcium, magnesium, guanidine, citrate, perchlorate, nitrate, thiocyanate, fluoride, chloride, sulfate, carbonate, pyrophosphate, phosphate, carboxylate, and cosmotropic salts of combinations thereof.

9. where the cosmotropic salt is tetrasodium pyrophosphate (Na ４ P ２ O ７ ), potassium dihydrogen phosphate (KH ２ PO ４ ), dipotassium hydrogen phosphate (K ２ HPO ４ ), tripotassium phosphate (K ３ PO ４ ), ammonium sulfate ((NH ４ ) ２ SO ４ ), sodium chloride (NaCl), sodium citrate monohydrate (NaC ６ H ７ O ７ ), disodium citrate (Na ２ C ６ H ６ O ７ ), trisodium citrate (Na ３ C ６ H ５ O ７ ), or a combination thereof, the method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 9, wherein step (a) is carried out under hydrophobic interaction chromatography (HIC) conditions.

11. The method according to claim 10, wherein the HIC conditions include the presence of a cosmotropic agent at a concentration of at least 0.01 M, and / or an electrical conductivity of at least 5 mS / cm, and / or a pH in the range of pH 4 to pH 10.

12. The method according to any one of claims 1 to 10, wherein the eluent used in step (c) is water, a solution that does not contain a cosmotropic agent, a solution that contains a low concentration of the at least one cosmotropic agent used in step (a), or a buffer having a pH in the range of pH 4 to pH 9, wherein the buffer does not contain a cosmotropic agent or contains a low concentration of the at least one cosmotropic agent.

13. The method according to any one of claims 1 to 12, wherein the solution containing the LNP comprises one or more organic solvents in a concentration of about 10% (volume / volume) to about 80% (volume / volume).

14. The method according to any one of claims 1 to 13, wherein the nucleic acid is selected from the group consisting of messenger RNA (mRNA), self-amplified RNA (saRNA), trans-amplified RNA (taRNA), self-replicating RNA (srRNA), circular RNA (circRNA), guide RNA (gRNA), small interfering RNA (siRNA), and mixtures thereof.

15. The use of a chromatographic medium having convection properties in the purification of lipid nanoparticles (LNPs) for encapsulating nucleic acids, wherein the chromatographic medium is used in the presence of at least one cosmotropic agent.

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