Method for purifying lipid nanoparticles

Abstract

The present invention relates to a method for purifying lipid nanoparticles (LNPs) encapsulating nucleic acids, comprising the steps of: subjecting a solution containing said LNPs to a chromatography medium having convective properties in the presence of at least one lyophilic agent; and eluting the LNP from the chromatography medium. The invention also relates to the corresponding use of a chromatography medium with convective properties in the purification of lipid nanoparticles (LNPs) encapsulating nucleic acids.

Description

[0001] The present invention relates to a method for purifying lipid nanoparticles (LNPs) encapsulating nucleic acids, comprising the steps of: subjecting a solution containing the LNPs to a chromatography medium having convection properties in the presence of at least one kosmotropic agent; and eluting the LNPs from the chromatography medium. The present invention also relates to the corresponding use of a chromatography medium having convection properties in purifying lipid nanoparticles (LNPs) encapsulating nucleic acids.

[0002] Nucleic acid-encapsulated LNPs have emerged as promising preventive and / or therapeutic agents, for example as vaccines carrying messenger RNA (mRNA), self-amplifying RNA (saRNA), or circular RNA (circRNA). LNPs are typically composed of ionizable cationic lipids, cholesterol, helper lipids, and PEGylated lipids and typically have a diameter of 30 to 300 nm, for example, 80 nm. However, LNPs are typically highly shear-sensitive.

[0003] The generation of mRNA such as sealing as vaccine is made up of multiple unit operations, comprise that microbial plasmid produces, plasmid separation, linearization, in vitro transcription reaction is to produce mRNA, it is by precipitation, chromatography or tangential flow filtration (TFF) purifying, and prepares under low conductivity buffer and gentle acidic conditions.Then mRNA is encapsulated in lipid nanoparticle, and described lipid nanoparticle is the spherical mixture of mRNA and ionizable lipid, obtains the particle of diameter about 100nm.By in microfluidic chamber with the lipid being dissolved in one or more organic solvents such as ethanol, methanol, acetone, isopropanol, ethyl acetate etc., carry out high pressure series mixing and realize mRNA is formulated as LNP.

[0004] The LNP produced like this is initially prepared in a high percentage of organic solvent (usually in the range of 10 to 50% (v / v)), which is harmful to the stability of nanoparticles and can change their size. Therefore, it is necessary to remove the organic solvent from the preparation by dilution or buffer exchange. This is usually achieved by TFF, usually with a molecular weight cut-off of 30kDa to 300kDa. However, this method produces high shear force and turbulence, which causes the degradation of LNP, thereby causing the low recovery of buffer-exchanged LNPs. In addition, because the cut-off size is less than the usual size of mRNA, unencapsulated mRNA is co-purified and concentrated together with LNP.

[0005] Therefore, the technical problem underlying the present invention is to provide a method for purifying nucleic acid-encapsulating LNPs with increased purity and yield.

[0006] The solution to the above technical problem is achieved through the embodiments described in the claims.

[0007] Specifically, in a first aspect, the present invention relates to a method for purifying lipid nanoparticles (LNPs) encapsulating nucleic acids, comprising the following steps:

[0008] (a) subjecting a solution containing the LNPs to a chromatography medium having convective properties in the presence of at least one kosmotropic agent;

[0009] (b) washing the chromatography medium with a solution containing at least one kosmotropic agent; and

[0010] (c) eluting the LNPs from the chromatography medium.

[0011] As described above, LNPs are nanoparticles typically composed of ionizable cationic lipids, cholesterol, helper lipids, and PEGylated lipids, typically having a diameter of 30 to 300 nm. However, the LNPs that can be purified in the methods of the present invention are not particularly limited and include all LNPs that may be of interest for the delivery / administration of nucleic acids.

[0012] Likewise, the nucleic acid encapsulated in the LNP is not particularly limited, and is included in any nucleic acid that may be interested in prevention and / or treatment environment. Corresponding nucleic acid includes single-stranded or double-stranded RNA and DNA molecules of any length, as a single species or a combination of two or more species, including such as messenger RNA (mRNA), self-amplification RNA (saRNA), trans-amplification RNA (taRNA), self-replicating RNA (srRNA), circular RNA (circRNA), guide RNA (gRNA), small interfering RNA (siRNA) and mixtures thereof, wherein gRNA, mRNA, saRNA and / or circular RNA are particularly preferred. The nucleic acid encapsulated in the LNP can also include any combination of two or more encapsulated nucleic acids, such as gRNA and mRNA, DNA and mRNA etc.

[0013] In step (a) of the method of the present invention, a solution containing LNPs is subjected to a chromatography medium having convective properties in the presence of at least one kosmotropic agent.

[0014] As used herein, the term "chromatographic medium with convective properties" refers to a chromatographic medium suitable for convective mass transfer, i.e., transporting a solution containing LNPs by convection, such as by gravity or by a pump, and which is not subject to diffusive mass transfer. Preferably, the chromatographic medium with convective properties has a low hydrophobicity, more preferably, in the presence of the at least one lyophile, which allows the LNPs to bind to the chromatographic medium but does not allow impurities, such as contaminating nucleic acids, that may be present in the solution containing the LNPs to bind to the chromatographic medium. Suitable materials for chromatographic media 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 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. The chromatographic medium can be functionalized with hydroxyl, C4, C6, C8, C12, C18, phenyl, or other ligands that are hydrophobic in the presence of a lyophilic salt. Each chromatography medium can be in the form of a chromatography support with convective properties, such as a block, a membrane, a nanofiber having a pore size of at least 0.3 μm up to 6 μm, preferably 1 to 2 μm, and a porous particle having a pore size and / or channel size of at least 0.3 μm up to 10 μm, preferably about 1 μm. In a preferred embodiment, the chromatography medium is a block chromatography medium functionalized with hydroxyl ligands. Thus, the chromatography medium can be a block chromatography medium, a membrane, a nanofiber, a porous particle as defined above, or any other chromatography device with convective properties of the solute.

[0015] The kosmotropic agent used in step (a) of the method of the present invention is preferably selected from the group consisting of kosmotropic salts of tetramethylammonium, ammonium, potassium, sodium, cesium, lithium, calcium, magnesium, guanidine, citrate, perchlorate, nitrate, thiocyanate, fluoride, chloride, sulfate, carbonate, phosphate including pyrophosphate, carboxylates, and combinations thereof. More specifically, the kosmotropic salt can be tetrasodium pyrophosphate (Na4P2O7), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), tripotassium phosphate (K3PO4), ammonium sulfate ((NH4)2SO4), sodium chloride (NaCl), monosodium citrate (NaC6H7O7), disodium citrate (Na2C6H6O7), trisodium citrate (Na3C6H5O7), or combinations thereof.

[0016] The present invention utilizes a chromatography medium having convective flow properties in the presence of at least one lyophile, i.e., under specific hydrophobic interaction chromatography (HIC) conditions, wherein these conditions enable selective binding of LNPs to the medium. Such HIC conditions include the presence of a lyophile at a concentration of at least 0.01 M, preferably at least 0.05 M, more preferably 0.1 to 1 M, more preferably 0.3 to 0.5 M, for example about 0.4 M. In addition, such HIC can also include a conductivity of at least 5 mS / cm and / or a pH of 4 to pH 10, preferably pH 7 to pH 9, more preferably pH 7.4 to pH 8.0.

[0017] In a particular embodiment, step (a) of the process of the present invention is preferably carried out at ambient temperature, for example at a temperature of about 20°C to about 25°C.

[0018] In step (b) of the process of the invention, the chromatography medium is washed with a solution comprising at least one kosmotropic agent.

[0019] The kosmotropic agent used in step (b) of the method of the present invention is preferably selected from the group consisting of kosmotropic salts of tetramethylammonium, ammonium, potassium, sodium, cesium, lithium, calcium, magnesium, guanidine, citrate, perchlorate, nitrate, thiocyanate, fluoride, chloride, sulfate, carbonate, phosphate including pyrophosphate, carboxylates, and combinations thereof. More specifically, the kosmotropic salt can be tetrasodium pyrophosphate (Na4P2O7), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen 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 kosmotropic agent used in step (b) of the method of the present invention is the same kosmotropic agent used in step (a) of the method of the present invention.

[0020] Furthermore, the solution containing at least one lyophile used in step (b) of the method of the present invention preferably comprises a lyophile in a concentration of at least 0.01 M, preferably at least 0.05 M, more preferably from 0.1 to 1 M, more preferably from 0.2 to 0.3 M. Furthermore, the solution can have a conductivity of at least 5 mS / cm and / or a pH of from pH 4 to pH 10, preferably from pH 7 to pH 9, more preferably from pH 7.4 to pH 8.0. Preferably, in the solution, the concentration and / or conductivity and / or pH of the lyophile is the same as the concentration and / or conductivity and / or pH of the lyophile used in step (a) of the method of the present invention.

[0021] In a specific embodiment, step (b) of the process 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 this context, the temperature is preferably the same as in step (a) of the process of the present invention.

[0022] In step (c) of the inventive method, the LNPs are eluted from the chromatographic medium, preferably by reducing the concentration of a lyophile. Preferably, the eluent used in this step is water, a solution containing no penetrant and no low concentration of the at least one lyophile used in step (a), or a buffer having a pH of between pH 4 and pH 9, such as a Tris buffer, containing no lyophile and no low concentration of the at least one lyophile. If present, such elution may comprise the presence of a lyophile at a concentration of at least 0.0001 M, preferably 0.0005 to 0.002 M, more preferably about 0.001 M.

[0023] In a specific embodiment, the solution containing LNPs used in the method of the present invention comprises an organic solvent derived from a previously described LNP production method, such as ethanol. Thus, the method of the present invention can further comprise, prior to step (a), a step of diluting the solution containing the LNPs in a solution containing the at least one kosmotropic agent. For example, prior to dilution, the solution containing the LNPs can have an organic solvent concentration of typically about 10% (v / v) to about 80% (v / v), preferably about 10% (v / v) to about 50% (v / v), wherein after dilution, the diluted solution has an organic solvent concentration of 10% (v / v) or less.

[0024] In a second aspect, the present invention relates to the use of a chromatography medium having convective properties for purifying lipid nanoparticles (LNPs) encapsulating nucleic acids, wherein the chromatography medium is used in the presence of at least one kosmotropic agent.

[0025] In this respect, all relevant limitations defined for the first aspect of the invention also apply to the second aspect of the invention. In particular, the chromatography medium having convective properties, the LNP, the nucleic acid and the kosmotropic agent are as defined above.

[0026] As used herein, the term “comprising” expressly includes the terms “consisting essentially of” and “consisting of”, ie, all said terms are interchangeable with each other herein.

[0027] In addition, as used herein, the term "about" preferably refers to a variation of ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.5% of a particular value. Thus, for example, the term "about 100" can include 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.

[0028] The present invention provides purification methods that improve the yield and purity of LNPs encapsulating nucleic acids such as mRNA.

[0029] Specifically, the present invention provides a method for concentrating, buffer-exchanging and purifying LNP in a single step. Convective chromatography media such as bulk, membrane and nanofiber are composed of interconnected flow channels functionalized with ligands. Porous particles with macropores (pore size or channel size greater than 0.3 μm) are also considered to be convection chromatography media. Due to the laminar flow properties of the mobile phase flowing through the interconnected channels or macropores, analyte is exposed to relatively low shear forces compared to TFF. By appropriately selecting ligands, selectivity for target analytes can be achieved under low shear conditions.

[0030] The present invention chemically combines a low shear environment with a ligand that binds to LNPs but not to mRNA or other free nucleic acid chains to achieve concentration, purification, and buffer exchange of LNPs after encapsulation.

[0031] The present invention uses a chromatographic medium with convective properties and low hydrophobicity under hydrophobic interaction chromatography (HIC) conditions to bind LNPs in the presence of a lyophilic salt such as potassium phosphate and elute them with a low concentration of the lyophilic salt in the presence of a buffer or water, thereby achieving buffer exchange. The LNPs formulated in a solution with a high concentration of organic solvent (i.e., the product of the encapsulation step, for example, having an organic solvent concentration of about 10% (v / v) to about 80% (v / v)) are first diluted in a mobile phase containing a lyophilic salt and applied to a chromatographic device in the same mobile phase at ambient temperature. Due to the convective properties of the chromatographic medium, the binding capacity of the LNPs is independent of the flow rate; therefore, the flow rate during loading can be high and the loading time short; therefore, the high initial dilution of the LNPs required for dilution of the organic solvent in the mixture is not limiting. In another embodiment, the LNP formulated in a solution with a high concentration of an organic solvent (i.e., the product of the encapsulation step) is first diluted in a low conductivity matrix (e.g., PBS or Tris buffer) and is formulated into a loading buffer by serial dilution and then immediately loaded onto the chromatographic medium. As in the previous embodiment, the dilution of LNP can be high without affecting purification time or efficiency. The hydrophobic matrix (e.g., hydroxyl) of the chromatographic medium keeps hydrophobic LNP during loading. However, due to the lower hydrophobicity of the contaminated mRNA or other unencapsulated nucleic acids compared with LNP, the former is not retained by the chromatographic unit. The LNP is then eluted into a low conductivity buffer or water (e.g., in a gradient or step) and then washed with water. Figure 1 Due to the convective nature of the chromatographic medium, the elution of LNPs is not controlled by diffusion limitations, which would result in broadening of the chromatographic peaks; a small amount of eluent (e.g., 3 to 5 column volumes) is required to achieve elution from the chromatographic medium, which results in a high concentration of LNPs in the eluted fraction, thereby concentrating the LNPs.

[0032] The feature of the chromatographic medium with convection properties of application has multiple benefits for the economy and efficiency of the downstream purification of LNP.First, compared with the method used in this area, by minimizing LNP exposure to destructive shearing forces, the productive rate of LNP production has been improved.Secondly, compared with the method used in this area, by the chemical differentiation of target analyte and impurity (such as mRNA), not currently realized by the method used in this area (such as TFF, it can not distinguish LNP and nucleic acid chain based on molecular weight), it has increased purification factors.This has increased the purity of the final product, thereby increased clinical efficacy and reduced the immunogenicity from residual mRNA.Finally, method of the present invention can be extended to industrial (multi-gram) production scale.

[0033] The value of this technology can be very high. The mRNA or other nucleic acid chains of LNP encapsulation are increasingly used as independent vaccines for multiple indications. The production of LNP is in high demand, so there is pressure to improve the productive rate of production / purification process. The methods used in the prior art are suboptimal because they cause the destruction of particles and can not remove key impurities. By applying the methods described herein, the LNP process yield and product purity can be improved, thereby improving the overall process recovery.

[0034] The accompanying drawings show:

[0035] Figure 1 ：

[0036] Schematic diagram of purification of nucleic acid-encapsulated lipid nanoparticles (LNPs) free of contaminating 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 lyophile; 2) A wash step elutes free RNA, residual organic solvent, and lipids from the column, while the LNPs remain bound; 3) Switching the eluent to water (or a solution containing a low concentration of the at least one lyophile or a buffer at pH 4 to 9) releases the LNPs from the chromatography medium.

[0037] Figure 2:

[0038] A: Preparative chromatogram of LNP purification using CIMmultus OH blocks with potassium phosphate as the lyophilic salt (1 mL bed volume, 2 mm channel diameter). 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). Loading, washing, and elution fractions are shown. B: Zoom-in of the loading stage of LNP purification on CIMmultus OH blocks with potassium phosphate as the lyophilic 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 the 260 and 280 nm signals, but not MALS, was observed, indicating breakthrough of free (unencapsulated) mRNA.

[0039] Figure 3 ：

[0040] Analytical reversed-phase (CIMac SDVB) chromatograms: i) mRNA standard, ii) LNP standard, iii) flow-through fraction (FT) from CIMmultus OH purification of LNP preparation, iv) elution fraction (E) from CIMmultus OH purification. The flow-through fraction shows the presence of material with a retention time of approximately 2.5 min, consistent with the elution profile of mRNA; the elution fraction shows the presence of material with a retention time of approximately 6.9 minutes, consistent with the elution profile of LNP.

[0041] Figure 4 ：

[0042] Preparative chromatogram of LNP purification using CIMmultus OH block (1 mL bed volume, 2 μm channel diameter) with sodium citrate as the kosmotropic salt as described in Example 2. Trace A: UV absorbance at 260 nm, Trace B: Multi-angle light scattering (MALS). Loading, washing, and elution fractions are shown.

[0043] Figure 5 ：

[0044] Analytical reverse phase chromatography of loading, flow-through (FT), and elution fractions (E) from the CIMmultus OH purification of the LNP formulation described in Example 2. The flow-through fraction showed the presence of material with a retention time of approximately 2.8 min, consistent with the elution profile of mRNA; the elution fraction showed the presence of material with a retention time of approximately 7.1 minutes, consistent with the elution profile of LNP.

[0045] Figure 6 ：

[0046] Preparative chromatogram of LNP purification using CIMmultus OH block (1 mL bed volume, 2 μm channel diameter) with serial dilution of LNPs into sodium citrate as the kosmotropic salt, as described in Example 3, wherein: Trace A: UV absorbance at 260 nm, Trace B: Multi-angle light scattering (MALS). Loading, washing, and elution fractions are shown.

[0047] Figure 7 ：

[0048] Analytical reversed-phase chromatograms of the loading, flow-through (FT), and elution fractions (E) obtained from the CI 1 Mmultus OH purification of loaded LNPs by the serial dilution procedure described in Example 3. The flow-through fraction shows the presence of material with a retention time of approximately 2.8 minutes, consistent with the elution profile of mRNA; the elution fraction shows the presence of material with a retention time of approximately 7.1 minutes, consistent with the elution profile of LNPs.

[0049] Figure 8 ：

[0050] Nanoparticle tracking analysis (NTA), as described in Example 3, shows the particle size distribution of eluted fractions a) loaded and b) purified using CIMmultus OH blocks with serial dilution of the LNPs into sodium citrate as the kosmotropic salt.

[0051] Figure 9 ：

[0052] Preparative chromatogram of LNP purification using CIMac™ OH block (0.1 mL bed volume, 6 mm channel diameter) with sodium citrate as the kosmotropic salt. Dotted trace: UV absorbance at 260 nm, dashed trace: UV absorbance at 280 nm, solid trace: multi-angle light scattering (MALS). Loading, elution, and clean-in-place fractions are shown. Chromatograms were acquired using a PATfixR LC system, and data acquisition and analysis were performed using PATfixR software. Preparation and purification as described in Example 4.

[0053] Figure 10 ：

[0054] Analytical chromatogram of an LNP sample from a 2D chromatography, showing the ion-pair reversed-phase chromatography portion. Dotted trace: UV absorbance of blank at 260 nm, dashed trace: UV absorbance of crude LNP preparation at 260 nm, solid trace: UV absorbance of eluted LNP sample at 260 nm. The first peak represents naked mRNA eluted using increasing acetonitrile concentrations. The second peak represents encapsulated mRNA eluted using increasing acetonitrile concentrations. Chromatograms were acquired using a PATfixR LC system, and data acquisition and analysis were performed using PATfixR software. Preparation and purification are described in Example 4.

[0055] Figure 11 ：

[0056] A: shows sample size and concentration analysis from a Myriade Videodrop measurement of the crude LNP formulation described in Example 4. B: shows sample size and concentration analysis from a Myriade Videodrop measurement of the eluted LNP sample described in Example 4. This shows that the size remains approximately the same while the concentration increases (necessitating recalculation due to dilution, which is described in Example 4).

[0057] Figure 12:

[0058] Cryo-TEM analysis of LNPs, A: main elution sample from Example 4, B: control particles, buffer exchanged only after formulation, C: flow-through of preparative chromatography. It shows no significant change in particles on the OH column, while only particles with low electron density were observed in the flow-through, indicating empty particles. Analysis was performed on a JEM-1230TEM (JEOL) microscope with a Gatan Ultrascan 4000SP CCD detector. Samples were prepared by distributing them on L-polylysine grids and then immersing them in liquid ethane.

[0059] Figure 13 ：

[0060] Preparative chromatogram of LNP purification using CIMac™ C4 block (0.1 mL bed volume, 2 mm channel diameter) with sodium citrate as the lyophilic salt. The traces are described in brackets in the figure legend. The broad peak in the MALS trace is the elution peak, which decreases with decreasing conductivity. The sharp peak following the elution peak is the clean-in-place peak. The peak at the beginning of the chromatogram with high conductivity indicates the flow-through fraction, and the UV trace clearly shows free mRNA (ratio UV 260 / 280 = 2). The chromatogram was obtained using a PATfixR LC system, and data acquisition and analysis were performed using PATfixR software. Preparation and purification described in Example 5.

[0061] Figure 14 ：

[0062] Preparative chromatogram of LNP purification using Poros 50OH resin and sodium citrate as the kosmotropic salt by the serial 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). Loading, washing, and elution fractions are shown.

[0063] Figure 15:

[0064] Nanoparticle tracking analysis (NTA), as described in Example 6, shows a) loading and b) size distribution of eluted fractions of LNP purification using Poros OH resin with serial dilution of the LNPs into sodium citrate as the kosmotropic salt.

[0065] The present invention will be further illustrated by the following examples, but is not limited thereto. Example

[0066] Example 1:

[0067] Purification of LNPs using CIMmultus OH in potassium phosphate

[0068] The HPLC-MS / MS was used according to the manufacturer's protocol (N / P ratio 4, flow rate ratio of aqueous solution to lipid solution 3:1, total flow rate 12 mL / min, final sample volume 0.5 mL). Ignite TM Approximately 100 μg of mRNA encoding eGFP (995 nucleotides) (0.2 mg / mL starting concentration) was encapsulated into lipid nanoparticles using a Genvoy ILM lipid mix. The LNP sample was directly diluted in 1 M potassium phosphate (total volume 12 mL) and loaded onto a CIMmultus OH (1 mL, 2 μm pore size). During sample loading, a gradual increase in UV260 / 280 nm signal was observed, but no MALS signal was observed ( Figure 2B After sample loading, the column was washed with mobile phase A (1 M potassium phosphate) until the UV signal returned to baseline. Elution was performed in a single step with 100% ddH2O. A significant increase in UV 260 / 280 nm and MALS signal was observed.

[0069] By reverse phase HPLC ( Figure 3 ) Analysis of samples corresponding to the flow-through and elution fractions showed the presence of RNA, but not LNPs, in the flow-through and elution fractions, and the presence of LNPs, but not RNA, in the elution fraction.

[0070] Example 2:

[0071] Purification of LNPs using CIMmultus OH in sodium citrate

[0072] 0.2 mg of mRNA encoding eGFP (995 nucleotides) was encapsulated according to the above method. The LNP product (1.6 mL) was immediately diluted 10-fold in PBS to reduce the ethanol concentration to <3%. 15 mL of the sample was prepared for loading by diluting it in mobile phase A (15 mM Tris, 250 mM sodium citrate, pH 7.51) to match MPA conductivity and pH. After loading the sample, the column was washed with 10 mL of MPA. Elution was performed by steps from 100% MPA to 100% MPB (15 mM Tris, pH 7.52); 100% MPB was kept for 10 mL (10 column volumes). Deionized water (dH2O; 10 mL) was used for column banding and the column was disinfected using 1 M NaOH (5 column volumes). Chromatographic separation was performed as shown in FIG. Figure 4As shown. A slight increase in the MALS signal between 15 and 20 mL indicates breakthrough of the LNP (exceeding the binding capacity). A strong increase in UV 260 and MALS signals was observed upon elution in MPB (30 mL). Reversed phase chromatography analysis confirmed the presence of free (unencapsulated) mRNA in the flow-through, but not in the elution fraction, and the presence of LNP in the elution fraction (E), but not in the flow-through ( Figure 5 Ribogreen analysis of the fractions (comparison of the Ribogreen fluorescence signal of RNA before and after detergent treatment to release the encapsulated RNA) showed that the LNP elution recovery was at least 60%. Nanoparticle tracking analysis showed the presence of particles with diameters ranging from 140 to 185 nm, confirming the presence of LNPs in the CIMmultus OH elution.

[0073] Example 3;

[0074] Purification of LNPs using serial dilution of CIMmultus OH in sodium citrate

[0075] The HPLC-MS / MS was used according to the manufacturer's protocol (N / P ratio 6, flow rate ratio of aqueous and lipid solutions 3:1, total flow rate 12 mL / min, final sample volume 1.6 mL). Ignite TM , about 0.2mg 4000 nucleotide mRNA (0.1mg / mL starting concentration) is encapsulated into lipid nanoparticles using Genvoy ILM lipid mixture. LNP product (1.6mL) is diluted 10 times in PBS immediately to reduce the ethanol concentration to <3%. 15mL of this sample is loaded with serial dilution to match the conductivity and pH of MPA (15mM Tris, 250mM sodium citrate, pH 7.51) using dilution buffer (30mMTris, 500mM sodium citrate, pH 7.5). After loading the sample, 6mL MPA washing column is used. Elution is carried out by a step from 100% MPA to 100% MPB (dH2O); 100% MPB is kept for 12mL (12 column volumes). 1M NaOH (5 column volumes) is used to disinfect the column. Chromatographic separation is as follows Figure 6 As shown. A strong increase in UV260 and MALS signals was observed at 100% MPB (elution), indicating the elution of LNPs. Reverse phase chromatography analysis confirmed a low signal corresponding to free (unencapsulated) mRNA in the flow-through but not in the elution fraction, as well as the presence of LNPs in the elution fraction (E) but not in the flow-through (FT). Figure 7Ribogreen analysis of the fractions (comparison of the Ribogreen fluorescence signal of RNA before and after detergent treatment to release the encapsulated RNA) showed that the LNP elution recovery was 95%. Tracking analysis of the loaded nanoparticles showed a broad size distribution with a mode value of 201 nm and a loading concentration of 4.4 × 10 9 particles / mL( Figure 8 a). The eluted fraction (E) showed the presence of particles with a modal diameter of 142 nm ( Figure 8 b); the particle concentration is 4.5×10 10 particles / mL, confirming the presence of LNPs and a 10-fold concentration of particles relative to loading.

[0076] Example 4:

[0077] Purification of LNPs using CIMac OH serial dilution in sodium citrate

[0078] use Ignite TM , 4000 nucleotide mRNA (128 μ g, 170 μ g / mL, in 100mM NaOAc, pH 4.0) is encapsulated in lipid nanoparticles.Aqueous stream and lipid solution stream (molar ratio: ALC-0315: DSPC: PEG-2KDMG: cholesterol=9.1: 1.8: 1.0: 8.9; 12.5mM EtOH) with flow rate ratio is 3: 1, and total flow rate is that 2mL / min mixes.LNP product (0.8mL) is diluted 10 times in PBS immediately so that ethanol concentration is reduced to <3%.By using dilution buffer (20mM Tris, 250mM sodium citrate, pH 7.4) serial dilution with the flow velocity of 10CV / min load 5mL this sample to match the conductivity and pH of MPA (15mM Tris, 200mM sodium citrate, pH 7.4). After loading the sample, the column was washed with 1 mL of MPA. Elution was performed by steps from 100% MPA to 100% MPB (15 mM TRIS, pH 7.4); 100% MPB was maintained for 5 minutes (50 column volumes). The column was disinfected with 1 M NaOH (20 column volumes). Chromatographic separation was performed as shown in FIG. Figure 9 As shown. 100% MPB (elution) was observed, i.e., a strong increase in UV260 and MALS signals was observed, indicating the elution of LNPs. Ion pair reversed phase chromatography analysis demonstrated that the percentage of encapsulated mRNA was much higher compared to the eluted fractions and crude samples ( Figure 10 Videodrop size analysis of the loading indicated the presence of a particle size distribution with a mode value of 115 nm and a loading concentration of 1.3 × 10 10 Particles / mL( Figure 11a). The eluted fraction (E) showed the presence of particles with a modal diameter of 115 nm ( Figure 11 b); the particle concentration is 3.3×10 10 The presence of LNPs and the concentration of particles were confirmed at 100 μg / mL. Analysis of the in-situ wash fraction showed the presence of very large particles (molten, agglomerated LNPs), while the flow-through fraction showed a very low concentration of particles (Figure 12c), primarily those with low electron density (voids). Cryo-TEM analysis confirmed that the eluted particles were mostly spherical (Figure 12a), had high electron density on average, and were morphologically comparable to control particles (buffer exchange only after formulation, Figure 12b).

[0079] Example 5:

[0080] Purification of LNPs in sodium citrate using C4 ligand-modified CIMac

[0081] LNPs were prepared from 4000 nucleotide mRNA and lipid mixture (molar ratio: SM-102: DSPC: PEG-2KDMG: cholesterol = 50: 10: 1.5: 38.5). Samples were prepared and loaded onto a column with buffer A (100 mM, 15 mM TRIS, pH 8.0). Elution was performed by a 30 CV gradient from 100% A to 100% buffer B (15 mM TRIS, pH 8.0) and maintained at 100% B for another 20 CV. The column was disinfected using 1 M NaOH (20 CV). Chromatographic separation was performed as shown in FIG. Figure 13 As shown. A flow-through peak was observed in the UV spectrum, corresponding to free mRNA from the UV 260 / 280 nm ratio. MALS peaks eluted during the elution gradient, and some MALS peaks were present in the disinfection phase. Those corresponded to LNPs.

[0082] Example 6:

[0083] Purification of LNPs by serial dilution in sodium citrate using POROS OH

[0084] According to the manufacturer's protocol (N / P ratio 4, flow rate ratio of aqueous and lipid solutions 3:1, total flow rate 12 mL / min, final sample volume 3.8 mL), Ignite TM, about 0.5 mg of 4000 nucleotide mRNA (0.2 mg / mL starting concentration) was encapsulated into lipid nanoparticles. The LNP product (3.8 mL) was immediately diluted 20-fold in PBS to reduce the ethanol concentration to <3%. 70.4 mL of the sample was loaded on Poros 50OH resin by serial dilution using dilution buffer MPA (30 mM Tris, 1.5 M sodium citrate, pH 7.5), of which 4.8 mL of wet sedimentation volume was filled in 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 to 100% MPC (dH2O) by steps; 100% MPC was maintained for 25 mL. The column was disinfected with 1 M NaOH 2M NaCl. Chromatographic separation was performed as follows Figure 14 As shown. 100% MPC (elution) was observed, i.e., a strong increase in UV 260 and MALS signals was observed, indicating the elution of LNPs. Ribogreen analysis of the fractions (comparison of Ribogreen fluorescence signals of RNA before and after detergent treatment to release the encapsulated RNA) showed that the LNP elution recovery was 78%. Tracking analysis of the loaded nanoparticles showed a broad size distribution with a mode value of 91 nm and a loading concentration of 9.08×10 10 The eluted fraction (E) showed the presence of particles with a modal diameter of 118 nm (Figure 15b); the particle concentration was 1.54 × 10 11 particles / mL, confirming the presence of LNPs.

Claims

1. A method for purifying lipid nanoparticles (LNPs) encapsulating nucleic acids, comprising the steps of: (a) subjecting a solution containing the LNPs to a chromatography medium having convective properties in the presence of at least one kosmotropic agent; (b) washing the chromatography medium with a solution containing at least one kosmotropic agent; as well as (c) eluting the LNPs from the chromatography medium.

2. The method of claim 1, wherein the chromatography medium is hydrophobic in the presence of the at least one lyophile, the lyophile allowing the LNP to bind to the chromatography medium but not allowing contaminating nucleic acids to bind to the chromatography medium.

3. The method of claim 1 or 2, wherein the chromatographic medium is a synthetic or natural organic polymer.

4. The method of any one of claims 1 to 3, wherein 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.

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

6. The method of any one of claims 1 to 5, wherein the chromatography medium is a bulk chromatography medium, a membrane, a nanofiber or a porous particle.

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

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

9. The method of any one of claims 1 to 8, wherein the kosmotropic salt is tetrasodium pyrophosphate (Na4P2O7), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), tripotassium phosphate (K3PO4), ammonium sulfate ((NH4)2SO4), sodium chloride (NaCl), monosodium citrate (NaC6H7O7), disodium citrate (Na2C6H6O7), trisodium citrate (Na3C6H5O7), or a combination thereof.

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

11. The method of claim 10, wherein the HIC conditions comprise the presence of a kosmotropic agent at a concentration of at least 0.01 M, and / or a 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 containing no kosmotropic agent and containing no low concentration of the at least one kosmotropic agent used in step (a), or a buffer having a pH between pH 4 and pH 9, the buffer containing no kosmotropic agent and containing no low concentration of the at least one kosmotropic agent.

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

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

15. Use of a chromatography medium with convective properties for purifying lipid nanoparticles (LNPs) encapsulating nucleic acids, wherein the chromatography medium is used in the presence of at least one kosmotropic agent.

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