Pharmaceutical composition comprising protein-bound nanoparticles and manufacturing method thereof

Abstract

A pharmaceutical composition contains protein-bound nanoparticles and 5 mM to 500 mM of arginine. The pharmaceutical composition may further contain 10 mM to 2 M of a disaccharide. The pharmaceutical composition may be in the form of a lyophilized formulation. The protein-bound nanoparticles may contain cationic lipids or ionizable cationic lipids, phospholipids, sterols, polyethylene glycol (PEG)-modified lipids, and conjugates of proteins and lipids

Description

NDJP.026WO PATENTPHARMACEUTICAL COMPOSITION COMPRISING PROTEIN-BOUND NANOPARTICLES AND MANUFACTURING METHOD THEREOFINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the PCT Request as filed with the present application are hereby incorporated by reference.SEQUENCE LISTING IN ELECTRONIC FORMAT

[0002] The present application is being filed along with an Electronic Sequence Listing in ST.26 format. The Electronic Sequence Listing is provided as an XML file named SL_NDJP_026WO.xml created and last saved on March 14, 2026, which is approximately 9.7 KB in size. The material in the Electronic Sequence Listing is incorporated herein by reference in its entirety in accordance with 35 U. S. C. § 1.52(e).FIELD

[0003] The present disclosure relates to a pharmaceutical composition comprising protein-bound nanoparticles, and manufacturing methods thereof.BACKGROUND

[0004] When delivering nucleic acids to target cells in the body, it is crucial to prevent their rapid degradation within the body. Additionally, the cellular uptake of nucleicNDJP.026WO PATENTacids presents significant challenges, making it necessary to develop methods for efficient intracellular delivery.

[0005] To address these issues, encapsulating nucleic acids in lipid nanoparticles has been proposed. This method not only protects nucleic acids from degradation but also takes advantage of the natural ability of lipid nanoparticles to be internalized by cells, enabling efficient intracellular delivery.

[0006] However, lipid nanoparticles are typically unstable in solution. As a result, pharmaceutical products containing lipid nanoparticles are often stored in a lyophilized form. During the lyophilization process, lipid nanoparticles are exposed to physical and chemical stresses that can compromise their stability.

[0007] U. S. Publication No. 10,300,018 discloses a technique to reduce changes in the particle size of lipid nanoparticles before and after lyophilization by adding cyclodextrins or sucrose to the suspension.

[0008] In recent years, significant progress has been made in developing techniques for efficiently delivering nucleic acids to target cells. For example, methods are being explored to conjugate antibodies or ligands with high affinity for antigens expressed on target cells to the surface of lipid nanoparticles. PCT Publication No. WO 2022 / 081694 discloses a method for delivering lipid nanoparticles to T cells by attaching anti-CD5 antibodies to their surfaces.

[0009] For lipid nanoparticles that carry protein components, storage through freezing or lyophilization is also considered a potential approach to prevent nucleic acid degradation. However, there has been insufficient investigation into suitable pharmaceuticalNDJP.026WO PATENTcompositions that can maintain the particle size stability of protein-carrying lipid nanoparticles under freezing or lyophilization conditions.SUMMARYProblems to be Solved by the Present Disclosure

[0010] Protein-bound nanoparticles encapsulating nucleic acids are composed of components with distinct physical properties, such as nucleic acids, proteins, and lipids. Due to their structural characteristics, these nanoparticles are susceptible to changes in particle size during processes such as freeze-thawing and lyophilization.

[0011] The present disclosure addresses these problems by providing a pharmaceutical composition that mitigates particle size changes in protein-bound nanoparticles during freeze-thawing or lyophilization. Furthermore, the present disclosure provides a manufacturing method designed to minimize such particle size changes in protein-bound nanoparticles.Means for Solving the Problems

[0012] The present disclosure adopts the following configurations to address the aforementioned issues. Specifically, the present disclosure includes the following embodiments [1] to [9]:[1] A pharmaceutical composition, comprising protein-bound nanoparticles and 5 mM to 500 mM of arginine.[2] The pharmaceutical composition according to [1] above, further comprising 10NDJP.026WO PATENTmM to 2 M of a disaccharide.[3] The pharmaceutical composition according to [1] or [2] above, wherein the protein-bound nanoparticles comprise cationic lipids or ionizable cationic lipids, phospholipids, sterols, polyethylene glycol (PEG)-modified lipids, and conjugates of proteins and lipids.[4] A pharmaceutical composition, comprising protein-bound nanoparticles and arginine, wherein a concentration of arginine in the pharmaceutical composition before a lyophilization is 5 mM to 500 mM, wherein the pharmaceutical composition is in a form of a lyophilized formulation.[5] The pharmaceutical composition according to [4] above, further comprising a disaccharide, wherein a concentration of the disaccharide in the pharmaceutical composition before a lyophilization is 10 mM to 2 M.[6] The pharmaceutical composition according to [4] or [5] above, wherein the protein-bound nanoparticles comprise cationic lipids or ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and conjugates of proteins and lipids.[7] A method of manufacturing a pharmaceutical composition, comprising:freezing protein-bound nanoparticles suspended in a freezing solution, wherein the freezing solution comprises 5 mM to 500 mM of arginine.[8] A method of manufacturing a protein-bound nanoparticle solution, comprising:mixing a nanoparticle solution and a protein solution to prepare a second mixedNDJP.026WO PATENTsolution; and performing a dialysis, a filtration, and / or a dilution of the second mixed solution using a freezing solution to obtain the protein-bound nanoparticle solution, wherein the freezing solution comprises 5 mM to 500 mM of arginine[9] The method according to [8] above, wherein the nanoparticle solution is obtained by subjecting a first mixed solution comprised of a first solution and a second solution to a dialysis, a filtration, and / or a dilution using a third solution, wherein the first solution comprises ethanol and at least a lipid having a chemically reactive functional group in its molecule, the second solution is an aqueous solution comprising at least a drug, and the third solution contains 5 mM to 500 mM of arginine.

[0010] The method according to [8] or [9] above, further comprising freezing the protein-bound nanoparticle solution after the dialysis, the filtration, and / or the dilution.

[0011] The method according to any one of [8]-

[0010] above, wherein the freezing solution further comprises 10 mM to 2 M of a disaccharide.

[0012] The method according to any one of [9]-

[0011] above, wherein the drug is a nucleic acid.

[0013] The method according to [9] above, wherein the chemically reactive functional group is a maleimide or an azide.

[0014] The method according to any one of [8]-

[0013] above, wherein: a pH of theNDJP.026WO PATENTfreezing solution is 6.5 to 8.5, a pH of the second solution is 3.0 to 7.0, and a pH of the third solution is 6.5 to 8.5.

[0015] The method according to any one of [8]-

[0014] above, wherein the first solution further comprises cationic lipids or ionizable cationic lipids, sterols, phospholipids, and PEG-modified lipids.

[0016] The method according to any one of [8]-

[0015] above, wherein the arginine is selected from the group consisting of L-Arginine, L-Arginine Hydrochloride, L- Arginine Acetate, L-Arginine Phosphate, L-Arginine Sulfate, D-Arginine, a- Arginine, N-acetylarginine, Arginine Ethyl Ester, Arginine Sodium, Arginine Monolaurate, L-Arginine Pyroglutamate, Succinylated Arginine, N- Methylarginine, Dimethylarginine, Arginine a-Ketoglutarate, and combination thereof.

[0017] The method according to any one of [8]-

[0015] above, wherein the arginine is selected from the group consisting of L-Arginine, L-Arginine Hydrochloride, L- Arginine Acetate, L-Arginine Phosphate, L-Arginine Sulfate, and combination thereof.

[0018] The method according to

[0011] above, wherein the disaccharide is selected from the group consisting of Sucrose, a-Trehalose, Leucrose, Murillosiose, Tricoccose, Melezitose, Thaumatinose, Lactosidol, Maltitol, Neotrehalose, Maltose, Lactose, Cellobiose, Trehalose, Isomaltose, Gentiobiose, Pleranose, Mannobiose, Laminaribiose, Sophorose, and combination thereof.

[0019] The method according to

[0011] above, wherein the disaccharide is Sucrose.NDJP.026WO PATENTEffects of the Present Disclosure

[0013] The pharmaceutical composition described in the present disclosure effectively reduces particle size changes in protein-bound nanoparticles caused by freezethawing or lyophilization. Additionally, the manufacturing method includes a step where protein-bound nanoparticles are contacted with arginine. This step enhances the stability of the particle size of protein-bound nanoparticles, protecting them from changes during freezing and thawing.DETAILED DESCRIPTION

[0014] The following illustrates modes for carrying out the present disclosure:(Pharmaceutical Composition)

[0015] The pharmaceutical composition of the present disclosure comprises protein-bound nanoparticles and arginine. The composition may take various forms, such as an aqueous solution, a frozen aqueous solution, or a lyophilized formulation. Among these, the frozen aqueous solution and lyophilized formulation forms are preferred for their ability to enable long-term storage of the composition.

[0016] The pharmaceutical composition preferably includes disaccharides, buffering agents, or metal chlorides. The protein-bound nanoparticles may preferably include a drug, cationic lipids or ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and protein-lipid conjugates.NDJP.026WO PATENT(Arginine)

[0017] Arginine is included in the pharmaceutical composition of the present disclosure to reduce particle size changes in protein-bound nanoparticles caused by freeze-thawing or lyophilization. Examples of arginine include: L-Arginine, L-Arginine Hydrochloride, L-Arginine Acetate, L-Arginine Phosphate, L-Arginine Sulfate, D-Arginine, a-Arginine, N-acetylarginine, Arginine Ethyl Ester, Arginine Sodium, Arginine Monolaurate, L-Arginine Pyroglutamate, Succinylated Arginine, N-Methylarginine, Dimethylarginine, and Arginine a-Ketoglutarate.

[0018] These may be used individually or in combination. Among these, L-Arginine, L-Arginine Hydrochloride, L-Arginine Acetate, L-Arginine Phosphate, and L-Arginine Sulfate are particularly preferred due to their identical structure to arginine utilized in the human body, indicating excellent safety for administration.

[0019] For aqueous solutions, the arginine concentration is preferably 5 mM to 500 mM, more preferably 5 mM to 200 mM, depending on the type and concentration of the protein-bound nanoparticles. A concentration of 5 mM to 200 mM is preferred for further reducing particle size changes caused by freeze-thawing.

[0020] For lyophilized formulations, the arginine concentration before lyophilization is preferably 5 mM to 500 mM, more preferably 5 mM to 250 mM. A concentration of 5 mM to 250 mM is particularly effective for reducing particle size changes caused by lyophilization.NDJP.026WO PATENT(Disaccharides)

[0021] The pharmaceutical composition of the present disclosure may further include disaccharides to more effectively reduce particle size changes caused by freeze-thawing or lyophilization. Examples of disaccharides include: Sucrose, a-Trehalose, Leucrose, Murillosiose, Tricoccose, Melezitose, Thaumatinose, Lactosidol, Maltitol, Neotrehalose, Maltose, Lactose, Cellobiose, Trehalose, Isomaltose, Gentiobiose, Pleranose, Mannobiose, Laminaribiose, and Sophorose.

[0022] These disaccharides may be used individually or in combination. Among these, Sucrose is particularly preferred for its superior ability to reduce particle size changes caused by freeze-thawing or lyophilization.

[0023] For aqueous solutions or frozen aqueous solutions, the concentration of disaccharides is preferably 10 mM to 2 M, more preferably 200 mM to 450 mM. A concentration of 200 mM to 450 mM is preferred for reducing particle size changes and aggregation caused by freeze-thawing or drying.

[0024] For lyophilized formulations, the disaccharide concentration before lyophilization is preferably 10 mM to 2 M, more preferably 200 mM to 450 mM. A concentration of 200 mM to 450 mM enhances the mechanical strength of the dried product, reducing damage during handling and transport.(Sucrose)

[0025] Examples of sucrose include Refined Sucrose, Crystalline Sucrose, Reduced Sucrose, Sulfonated Sucrose, Carboxylated Sucrose, and Phosphorylated Sucrose,NDJP.026WO PATENTas well as mixtures thereof. These may be used individually or in combination. Among these, Refined Sucrose and Crystalline Sucrose are particularly preferred due to their widespread use in food and pharmaceuticals and their excellent safety profiles.(Buffering Agents)

[0026] In addition to arginine, the pharmaceutical composition of the present disclosure may include buffering agents to maintain an appropriate pH range and ensure component stability. For structural stability of protein-bound nanoparticles, weak acid or weak base buffer systems are preferred.

[0027] Examples of buffering agents include phosphate buffers, Tris buffers, acetate buffers, and citrate buffers. The pH range and concentration of the buffering agent can be selected according to the intended purpose of the composition.

[0028] The concentration of the buffering agent is preferably 0.5 mM to 100 mM, but may be adjusted depending on the type of protein-bound nanoparticles and the formulation. A pH range of 6.5 to 8.5 is particularly preferred for preventing degradation or denaturation of the protein-bound nanoparticles.(Metal Chlorides)

[0029] The pharmaceutical composition of the present disclosure may further include metal chlorides in addition to arginine. Examples of metal chlorides include sodium chloride and potassium chloride, which may be used individually or in combination. The concentration of metal chlorides in the pharmaceutical composition of the present disclosure is preferably adjusted within a range of 0.01 M to 1 M. This range provides proper adjustmentNDJP.026WO PATENTof osmotic pressure while maintaining the physicochemical stability of the protein-bound nanoparticles. The choice of metal chlorides can be tailored to the intended application, but pharmaceutical-grade sodium chloride or potassium chloride is particularly preferred for biocompatibility and safety.(Protein-bound Nanoparticles)

[0030] In the present disclosure, protein-bound nanoparticles refer to structures in which at least one type of protein is bound to or immobilized on nanoparticles. These nanoparticles have a particle size (Z-average size) of 1 nm to 500 nm, preferably 10 nm to 200 nm, and more preferably 50 nm to 150 nm. This size range is desirable for applications in drug delivery, diagnostics, and therapeutics, providing effective distribution to target tissues and efficient intracellular uptake. The proteins used in the present disclosure include, but are not limited to, enzymes, antibodies, antibody fragments, peptides, and their derivatives. These proteins are utilized to modify the surface of nanoparticles, bind drugs, and / or impart biological functionality in vivo. The nanoparticles may be composed of lipids, inorganic materials, biocompatible polymers, or combinations thereof. When used fordrug delivery, nanoparticles can also encapsulate drugs. Examples of nanoparticle materials include lipids (cationic lipids, ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and protein-lipid conjugates), inorganic materials (silica, iron oxide, gold, silver, zinc oxide, titanium oxide, calcium phosphate, and carbon nanotubes), and biocompatible polymers (polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), PLA-PEG block copolymers, polyethylene glycol (PEG), polylysine, chitosan, dextran, hyaluronic acid, and alginate). These materials can be used individually or in combination to adjust the properties of the nanoparticles and expand their applications in pharmaceuticals or diagnostics. ProteinsNDJP.026WO PATENTin the protein-bound nanoparticles can bind to nanoparticles through covalent chemical bonds, electrostatic interactions, hydrophobic interactions, or non-covalent adsorption. Among these, covalent chemical bonds are preferred for their superior binding stability. Protein-bound nanoparticles can be used as drug delivery carriers or vaccine adjuvants. In particular, nanoparticles containing antibodies or antibody fragments are useful for targeted therapies in cancer and infectious disease treatment. For nucleic acid drug delivery, nanoparticles composed of cationic lipids, ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and protein-lipid conjugates efficiently encapsulate nucleic acids and facilitate their delivery to cells in vivo. Embodiments include nanoparticles formed with lipids (e.g., cationic lipids, ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids) and including nucleic acids as drugs, which can subsequently bind to antibodies via chemically reactive functional groups (e.g., on the nanoparticle surface), yielding protein-bound nanoparticles. Another embodiment involves forming nanoparticles with lipids (e.g., cationic lipids, ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, protein-lipid conjugates) and nucleic acids, resulting in protein-bound nanoparticles.(Particle Size)

[0031] The particle size (Z-average size) of nanoparticles and protein-bound nanoparticles in the present disclosure ranges from 1 nm to 500 nm, preferably from 10 nm to 200 nm, and more preferably from 50 nm to 150 nm. The Z-average size can be measured using dynamic light scattering (DLS) with an appropriate instrument such as a Malvern Zetasizer. For measurement, the particle suspension is appropriately diluted and injected into a measurement cell. Using the DLS mode, conditions such as temperature, viscosity, and refractive index are set, and the scattering light intensity data is analyzed to calculate the Z-NDJP.026WO PATENTaverage size based on the Brownian motion of the particles. This method also provides the polydispersity index (PDI), indicating the uniformity of particle size distribution.(Protein Concentration in the Pharmaceutical Composition)

[0032] When the pharmaceutical composition comprising protein-bound nanoparticles is in the form of an aqueous solution or frozen aqueous solution, the protein concentration is preferably 1 pg / mL to 10 mg / mL. This configuration significantly enhances nanoparticle size stability. For lyophilized formulations, the protein concentration in the composition before lyophilization is also preferably 1 pg / mL to 10 mg / mL, which significantly stabilizes nanoparticle size. The protein concentration or amount in the pharmaceutical composition can be measured using the BCA method (Pierce BCA Protein Assay Kit). Reagent A (BCA reagent) and Reagent B (alkaline copper reagent) are mixed at a 50:1 ratio to prepare the BCA working reagent. BSA (bovine serum albumin) standards are prepared in the range of 0 pg / mL to 400 pg / mL. The samples are diluted 10- to 100-fold with an appropriate buffer. 20 pL of the sample or standard solution is added to each well of a 96-well microplate, followed by 180 pL of the BCA working reagent. The plate is incubated at 37°C for 30 minutes, and the absorbance is measured at 562 nm. The protein concentration is calculated based on the standard curve.(Ionizable Cationic Lipids)

[0033] Ionizable cationic lipids of the present disclosure are lipids that can acquire a positive charge depending on the pH. They remain neutral at physiological pH and become positively charged in acidic environments. Examples of ionizable cationic lipids include DLin-MC3-DMA (1,2-dilinoleoyl-sn-glycero-3-methyldimethylamine), DODAP (1,2-dioleoyl-3-NDJP.026WO PATENTdimethylaminopropane), DDA (dimethyldioctadecylammonium), C12-200 (1,1'-[[(2- (dimethylamino)ethyl)imino]bis-bitanesanoil]ethylmethylamine), OF-Deg-Lin (oxyfluoroalkyl degrinol linoleic acid), ATLAS-75 (acylthio-lipid amine 75), DLin-KC2-DMA (1,2-dilinoleoyl-sn-glycero-3-dimethylaminoethanol), DLin-DMA (1,2-dilinoleoyl-sn-glycero-3-dimethylamine), ALC-0315 ([(4- hydroxy butyl) azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), SM-102 ([(4-hydroxybutyl) azanediyl]bis(hexane-6,1-diyl)bis(9-heptadecenate)), and compounds 1-3 represented by Formulas below.Compound 1:NDJP.026WO PATENTCompound 2:(Cationic Lipids)

[0034] Cationic lipids of the present disclosure are positively charged lipids. Examples include DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DC-Chol (3 -[N-(N', N'-dimethylaminoethyl)carbamoyl]cholesterol), DDAB (didodecyldimethylammonium bromide), DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane), DPTAP (1,2-dipalmitoyl-3-trimethylammonium-propane), DDA (dimethyldioctadecylammonium), and CTAB (cetyltrimethylammonium bromide). Cationic or ionizable cationic lipids can be used individually or in combination.NDJP.026WO PATENT(Phospholipids)

[0035] Phospholipids of the present disclosure are amphiphilic lipid molecules composed of hydrophobic tails and hydrophilic heads containing phosphate and polar groups. Examples include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOTMA (1,2-dioleoyl-3-trimethylammonium-propane), DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerin), DSPG (1,2-distearoyl-sn-glycero-3-phosphoglycerin), DPPG (1,2-dipalmitoyl-sn-glycero-3-phosphoglycerin), DOPA (1,2-dioleoyl-sn-glycero-3-phosphoamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate), DPPA (1,2- dipalmitoyl-sn-glycero-3-phosphate), and DSPA (1,2-distearoyl-sn-glycero-3-phosphate). Phospholipids may be used individually or in combination.(Sterols)

[0036] Sterols of the present disclosure are lipid molecules with a steroid backbone. Examples include cholesterol, epicholesterol, p-sitosterol, ergosterol, cholestanol, 7-dehydrocholesterol, androsterol, and desmethylcholesterol. Sterols may also be used individually or in combination.NDJP.026WO PATENT(PEG-Modified Lipids)

[0037] PEG-modified lipids of the present disclosure are amphiphilic molecules with polyethylene glycol (PEG) chains attached to their lipid molecules. Examples of PEG- modified lipids in the present disclosure include mPEG-DSPE (methoxy PEG-distearoyl phosphoethanolamine), mPEG-DMG (methoxy PEG-dimyristoylglycerol), mPEG-DOPE (methoxy PEG-dioleoyl phosphoethanolamine), mPEG-Ceramide (C14, C16, C18; methoxy PEG-ceramide), mPEG-Cholesterol (methoxy PEG-cholesterol), and mPEG-DSG (methoxy PEG-distearoylglycerol). These PEG-modified lipids may be used individually or in combination.(Lipids with Chemically Reactive Functional Groups)

[0038] The pharmaceutical composition of the present disclosure preferably employs lipids with chemically reactive functional groups. These lipids enable bonding with proteins and ensure efficient insertion of the hydrophobic domain into the nanoparticle lipid layer, while the reactive groups remain exposed on the surface, facilitating the formation of chemical bonds with proteins. This approach allows for the stable immobilization of proteins on nanoparticles and simplifies nanoparticle surface functionalization and stabilization.

[0039] Examples of chemically reactive functional groups include maleimide groups, which specifically react with thiol groups to form stable thioether bonds. For instance, maleimide groups in lipid molecules can react with thiol groups in proteins, resulting in a stable bond. Azide groups can react with alkyne groups via click chemistry (e.g., CuAAC reaction) to selectively attach specific molecules to the nanoparticle surface. Carboxyl groups react with amine groups to form amide bonds, providing high stability for drug components. AldehydeNDJP.026WO PATENTgroups react with amino groups to form Schiff bases, which can be reduced to achieve stable bonds.

[0040] Specific examples of such lipids include maleimide-PEG-DSPE, which improves nanoparticle stability and biocompatibility; azide-PEG-DSPE, which enables efficient binding via click chemistry; and carboxyl-PEG-DSPE, which forms stable amide bonds with amine groups. These lipids allow efficient bonding with proteins or therapeutic agents. For example, nanoparticles containing maleimide-PEG-DSPE can react with thiol groups in proteins to create protein-bound nanoparticles. This method operates under mild conditions, minimizing structural changes in proteins while achieving high binding efficiency.(Protein-Lipid Conjugates)

[0041] In one embodiment of the present disclosure, protein-lipid conjugates consist of lipid molecules chemically bonded to protein molecules. These conjugates are used as components of the pharmaceutical composition, contributing to surface modification of nanoparticles, imparting target specificity, and improving biocompatibility.

[0042] The bonding between proteins and lipids is achieved by introducing chemically reactive functional groups into lipid molecules. For instance, maleimide groups in lipids can react with thiol groups in proteins to form stable thioether bonds. This process minimizes effects on protein structure and activity under mild conditions. Azide groups in lipids can also react with alkyne groups in proteins via click chemistry (e.g., CuAAC reaction), achieving highly efficient and selective bonding.NDJP.026WO PATENT

[0043] Preferred lipid molecules include phospholipids with hydrophobic chains (e.g., DSPE or DPPC) conjugated to polyethylene glycol (PEG) and containing chemically reactive functional groups, facilitating protein immobilization on nanoparticle surfaces and enhancing nanoparticle stability and biocompatibility. Proteins include antibodies, antigenbinding fragments, enzymes, peptides, or derivatives thereof.

[0044] Specific examples include protein-lipid conjugates formed by reacting maleimide-PEG-DSPE with thiol groups in antibody molecules. These conjugates immobilize antibodies on nanoparticle surfaces, providing high binding affinity to specific cells or target molecules. Conjugates formed by reacting carboxyl-PEG-DSPE with amino groups in proteins via amide bonds are also feasible.(Drugs)

[0045] Drugs encapsulated in the lipid nanoparticles of the present disclosure include nucleic acids, small molecules, peptides, proteins, and other bioactive substances. Examples of nucleic acids include mRNA, siRNA, shRNA, DNA, antisense oligonucleotides, and plasmid DNA, which can regulate gene expression or inhibit target gene expression. Small molecule drugs include anticancer agents, antibiotics, antiviral agents, and anti¬ inflammatory agents. Encapsulation of these compounds in lipid nanoparticles enhances their stability and targeting specificity in vivo.

[0046] Examples of peptide and protein therapeutics include insulin, interferons, monoclonal antibodies, cytokines, and vaccine antigens. Encapsulation of these agents in lipid nanoparticles improves their stability and delivery efficiency. Other bioactive substances include vitamins, hormones, and immunomodulators encapsulated in lipid nanoparticles,NDJP.026WO PATENTbroadening the pharmaceutical applications of the composition. The lipid nanoparticles of the present disclosure can encapsulate a wide range of drugs, enabling diverse applications for the pharmaceutical composition.(mRNA)

[0047] mRNA of the present disclosure is an RNA molecule that carries genetic information serving as a template for protein synthesis within cells. It transfers genetic information transcribed from DNA to ribosomes, where specific proteins are synthesized. The structure of mRNA may include, but is not limited to, a cap structure, a 5' untranslated region (5' UTR), a coding region, a 3' untranslated region (3' UTR), and a poly-A tail. To reduce immunogenicity and enhance stability and translation efficiency, uridine in mRNA may be replaced with modified nucleotides such as 1-methyl-3'-pseudouridine or 5-methylcytidine.(siRNA)

[0048] siRNA of the present disclosure is an RNA molecule that functions in gene silencing to suppress the expression of specific genes. siRNA typically consists of double¬ stranded RNA (dsRNA) of 20-25 nucleotides with annealed sense and antisense strands, and may include a phosphate group at the 5' end. Chemical modifications, such as 2'- methoxyuridine, 2'-fluoro modifications, or phosphorothioate linkages, can be introduced to enhance siRNA stability, reduce immunogenicity, and improve target mRNA specificity.NDJP.026WO PATENT(miRNA)

[0049] miRNA of the present disclosure is a small non-coding RNA molecule, typically 20-22 nucleotides in length, that regulates gene expression by binding complementarily to target mRNA. It functions to suppress gene expression or modulate post- transcriptional regulation, thereby controlling protein expression. Chemical modifications such as 2'-O-methylation or the introduction of phosphorothioate linkages may be employed to enhance miRNA stability and binding efficiency to target mRNA.(Encapsulation Efficiency of Nucleic Acids)

[0050] In the present disclosure, encapsulation efficiency (EE) is defined as the percentage of nucleic acid encapsulated within nanoparticles relative to the total nucleic acid input during preparation. Encapsulation efficiency is calculated using the formula:EE (%) = ( Amount of nucleic acid in nanoparticles / Total amount of nucleic acid ) × 100Total amount of nucleic acid

[0051] To determine encapsulation efficiency, the nanoparticle sample is diluted with buffer, and the dilution factor is adjusted based on sample characteristics. Nonencapsulated nucleic acids are then separated using size-exclusion chromatography (SEC) or ultrafiltration (e.g., MWCO filters of 10 kDa-100 kDa). This process removes free nucleic acids outside the nanoparticles, isolating only the encapsulated nucleic acids.

[0052] The encapsulated nucleic acids are quantified using methods such as fluorescent dye assays (e.g., RiboGreen, SYBR Green), qPCR, UV absorbance at 260 nm, or HPLC. Similarly, the total nucleic acid amount is measured from the entire sample usingNDJP.026WO PATENTthe same methods. Encapsulation efficiency is then calculated based on these measurements.

[0053] In the present disclosure, an encapsulation efficiency of 80% or higher is preferred, with 90% or higher being particularly preferred. Achieving this range of encapsulation efficiency enhances the efficacy of nucleic acids and improves the delivery efficiency of the nanoparticles.(Manufacturing Method)

[0054] In the manufacturing method of the present disclosure, a second mixed solution (obtained by combining a nanoparticle solution and a protein solution under conditions selected to react a chemically reactive functional group on the lipid with the protein) is subjected to dialysis, filtration, and / or dilution using a freezing solution to prepare a protein¬ bound nanoparticle solution. The freezing solution contains 5 mM to 500 mM arginine.(Nanoparticle Solution)

[0055] In the manufacturing method of the present disclosure, the nanoparticle solution is prepared by subjecting a first mixed solution (comprising a first solution and a second solution) to dialysis, filtration, and / or dilution using a third solution. The first solution contains lipids with chemically reactive functional groups and a solvent such as ethanol. Optionally, the first solution may also include cationic lipids, ionizable cationic lipids, phospholipids, cholesterol, and PEG-modified lipids. The first solution preferably comprises these lipids dissolved in ethanol.NDJP.026WO PATENT

[0056] The second solution is an aqueous solution containing at least a drug and may optionally include buffering agents and / or metal chlorides. Nucleic acids can be used as the drug. The second solution is a solution in which the drug is dissolved in water. The pH of the second solution is preferably in the range of 3.0 to 7.0, with a range of 3.0 to 5.0 being particularly preferred.

[0057] The third solution is an aqueous solution containing 5 mM to 500 mM arginine and may optionally include buffering agents and metal chlorides. The pH of the third solution is preferably in the range of 6.5 to 8.5, with a range of 7.0 to 8.0 being particularly preferred.

[0058] The mixing of the first and second solutions is not particularly limited and can be carried out using various methods, such as:(i) simultaneous feeding of the first and second solutions from independent supply sources into a mixing apparatus, and / or(ii) adding the second solution to a container already containing the first solution.

[0059] Mixing parameters such as speed, time, and temperature can be controlled to achieve the desired mixing properties. Agitation devices may be used, with adjustable blade shapes, rotation speeds, and directions. Pre-heating or cooling of at least one of the solutions prior to mixing may also improve post-mixing properties. Further enhancements in mixing uniformity and reaction efficiency can be achieved through additional processes such as ultrasonication, pressurization, or vacuum treatment.NDJP.026WO PATENT

[0060] Continuous flow designs can be implemented where the two solutions merge just before a mixing point, enhancing process continuity. The mixing method can involve any single approach or a combination of these techniques.(Dialysis, Filtration, and Dilution of First Mixed Solution)

[0061] The first mixed solution obtained from the first and second solutions is preferably subjected to dialysis, filtration, and / or dilution using the third solution in the present disclosure. This allows arginine in the third solution to interact with nanoparticles, reducing particle size changes caused by freeze-thaw or lyophilization.Dialysis:

[0062] Dialysis removes unencapsulated nucleic acids and unwanted small molecules from the first mixed solution. The molecular weight cut-off (MWCO) of the dialysis membrane used in dialysis is appropriately selected based on the molecular weights of the components to be removed into the dialysate and those to be retained within the dialysis membrane. When retaining lipid nanoparticles or protein-bound lipid nanoparticles within the dialysis membrane, it is preferable to use a dialysis membrane with an MWCO of 10,000 to 100,000 Da and perform dialysis at 4°C or room temperature for approximately 12 to 48 hours. Additionally, replacing the buffer 2 to 3 times every 4 to 6 hours enables efficient removal of unwanted components. Using the third solution as the dialysis buffer enhances the stability of both nanoparticles and nucleic acids.NDJP.026WO PATENTFiltration:

[0063] Filtration is used to size-select the first mixed solution, remove aggregates, and improve uniformity. Ultrafiltration may be performed with 0.22 μm or 0.45 μm filters under low pressure (100–200 kPa) at 4°C or room temperature. This process efficiently removes aggregates and foreign matter while maintaining nanoparticle stability. Nanoparticles concentrated via ultrafiltration can be diluted with the third solution to bring arginine into contact with nanoparticles. Gel filtration with the third solution as the running buffer can also achieve similar results.Dilution:

[0064] Dilution adjusts the final concentration of nanoparticles and disperses them into the desired solution for use. Using the third solution during dilution facilitates arginine interaction with the nanoparticles, maintaining the stability of nanoparticles and nucleic acids.

[0065] By appropriately combining these processes, the manufacturing method of the present disclosure enables efficient production of highly stable protein-bound nanoparticles.(Protein Solution)

[0066] The protein solution in the present disclosure refers to a solution containing proteins, which include but are not limited to antibody solutions, antibody fragment solutions, enzyme solutions, peptide solutions, or ligand solutions. These can be used individually or in combinations of two or more. The protein components in the solution preferably contain thiol groups to facilitate reactions with maleimide groups, which are chemically reactive functionalNDJP.026WO PATENTgroups. Thiol groups enable conjugation with lipids containing chemically reactive functional groups in the nanoparticle solution. Consequently, in the second mixed solution obtained by combining the nanoparticle solution and the protein solution, the protein components bind to the nanoparticles, e.g., under conditions selected to react thiol groups of the protein with maleimide groups of the lipid.

[0067] The binding reaction between proteins and nanoparticles is preferably carried out under mild conditions at a pH range of 6.5-7.5 for several hours to overnight. These conditions preserve the activity of the proteins and allow efficient binding. After the reaction, unbound proteins are removed through additional filtration or dialysis, resulting in highly pure protein-bound nanoparticles.(Dialysis, Filtration, and Dilution of Second Mixed Solution)

[0068] In the pharmaceutical composition of the present disclosure, dialysis is performed to remove unbound proteins and low-molecular-weight impurities from the second mixed solution containing protein-bound nanoparticles. Dialysis may be conducted using dialysis bags or cartridges, with molecular weight cut-off (MWCO) membranes ranging from 10,000 to 100,000 Da, selected based on the properties of the protein-bound nanoparticles. The freezing solution is preferably used as the dialysis buffer to maintain the stability of the protein-bound nanoparticles. Dialysis is conducted at 4°C or room temperature for 12-48 hours, with buffer replacement 2-3 times every 4-6 hours.

[0069] Filtration may be employed to size-select the second mixed solution containing protein-bound nanoparticle, remove aggregates, and improve uniformity. Ultrafiltration using 0.22 μm or 0.45 μm filters may be conducted at low pressures of 100–200NDJP.026WO PATENTkPa under 4°C or room temperature, efficiently removing aggregates and foreign substances. Protein-bound nanoparticles concentrated through ultrafiltration may then be diluted using the freezing solution containing 5 mM to 500 mM arginine, allowing interaction with the proteinbound nanoparticles. When gel filtration is used, the freezing solution serves as the running buffer, enabling effective contact between arginine and the protein-bound nanoparticles.

[0070] Dilution is carried out to adjust the final concentration of protein-bound nanoparticles and disperse them into a solution suitable for the intended use. During dilution, it is highly preferable to maintain uniformity and stability of the protein-bound nanoparticles. Using the freezing solution for dilution in the manufacturing method facilitates effective interaction of arginine with the protein-bound nanoparticles. By appropriately combining dialysis, filtration, and dilution, the present disclosure enables efficient production of highly pure and stable protein-bound nanoparticles.(Freezing Solution)

[0071] The freezing solution in the present disclosure contains 5 mM to 500 mM arginine. This interaction of arginine with protein-bound nanoparticles effectively reduces particle size changes during freeze-thaw and lyophilization processes. Additionally, the freezing solution may include disaccharides in concentrations ranging from 10 mM to 2 M before lyophilization, which further suppresses particle size changes.

[0072] The freezing solution can also include buffer agents and metal chlorides. Buffer agents stabilize pH changes, enhancing nanoparticle stability, while metal chlorides contribute to osmotic pressure adjustment and surface charge stabilization.NDJP.026WO PATENT

[0073] While the pH of the freezing solution is not particularly limited, a range of 6.5-8.5 is preferred, with 7.0-7.5 being particularly favorable. This range maximizes the stability of the protein-bound nanoparticles and improves the performance of the pharmaceutical composition.(Protein-bound Nanoparticle Solution)

[0074] In the present disclosure, the protein-bound nanoparticle solution is obtained by subjecting the second mixed solution of the nanoparticle solution and the protein solution to dialysis, filtration, and / or dilution using the freezing solution.

[0075] This protein-bound nanoparticle solution is preferably frozen after dialysis, filtration, and / or dilution using the freezing solution. Additionally, the solution can be lyophilized after these processes, enhancing its stability and enabling long-term storage.(Frozen Storage Formulation)

[0076] The present disclosure provides a frozen storage formulation in which aqueous suspensions of protein-bound nanoparticles are preserved by freezing. The pharmaceutical composition of the present disclosure preferably includes 5 mM to 500 mM arginine to reduce particle size changes in protein-bound nanoparticles caused by freeze¬ thaw and freeze-drying processes. Additionally, combining 10 mM to 2 M disaccharides further enhances particle size stability. This allows for the maintenance of both the physical and chemical stability of protein-bound nanoparticles. The frozen storage formulation is particularly applicable to protein-bound nanoparticles containing nucleic acids and may include lipid components such as cationic lipids or ionizable cationic lipids, phospholipids,NDJP.026WO PATENTsterols, and PEG-modified lipids. These lipid components contribute to high-efficiency nucleic acid encapsulation, nanoparticle stability, and improved delivery efficiency within the body.

[0077] To maintain appropriate pH during storage, phosphate buffers, Tris buffers, or citrate buffers are preferably included. The pH range during storage is preferably between 6.5 and 8.5, which suppresses the degradation of nucleic acids and lipid components. The frozen storage formulation is characterized by a storage temperature of -20°C or lower, more preferably -80°C or lower, enabling long-term maintenance of particle size stability and biological activity of nucleic acids. This formulation is characterized by a particle size change of 80% to 120% after thawing.(Freezing Process)

[0078] The conditions for freezing and thawing suspensions of protein-bound nanoparticles are critical to maintaining nanoparticle structure and preserving drug activity, in the pharmaceutical composition of the present disclosure, rapid freezing is preferred for freezing suspensions of protein-bound nanoparticles. Rapid freezing minimizes structural changes in protein-bound nanoparticles and reduces nucleic acid denaturation. Specifically, freezing temperatures are set between -40°C and -80°C, and suspensions are frozen as quickly as possible to avoid damage caused by ice crystal formation. Particularly, rapid freezing using ultra-low temperatures (such as those of liquid nitrogen) is recommended to maintain nanoparticle uniformity.NDJP.026WO PATENT(Thawing Process)

[0079] Thawing conditions of the present disclosure should be gentle to avoid damaging the structure of protein-bound nanoparticles. Thawing is preferably conducted slowly at 4°C or room temperature to minimize physical stress caused by temperature fluctuations, thereby maintaining nanoparticle stability. When rapid thawing is necessary, thawing in a water bath at room temperature is feasible, but direct heating should be avoided to prevent abrupt expansion or contraction of nanoparticles. Under these conditions, the protein-bound nanoparticle suspension maintains its structure and drug activity after freezethaw processes, making it suitable for use as a stable pharmaceutical composition.(Lyophilized Formulation)

[0080] The present disclosure provides a lyophilized formulation enabling storage and transport by freeze-drying a liquid form of the pharmaceutical composition. The pharmaceutical composition of the present disclosure preferably includes 5 mM to 500 mM arginine to suppress particle size changes in protein-bound nanoparticles during freeze-drying and reconstitution. Combining 10 mM to 2 M disaccharides further enhances particle size stability, maintaining the physical and chemical stability of protein-bound nanoparticles.

[0081] To provide volume or mass and ensure sufficient physical strength of the dried formulation, excipients such as trehalose, mannitol, or sorbitol can be added. These excipients improve formulation uniformity and mechanical strength, promoting stability during freeze-drying and reconstitution.NDJP.026WO PATENT

[0082] In the lyophilized formulation, lipid components such as cationic lipids, ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and protein-lipid conjugates can be used. These lipids contribute to high-efficiency nucleic acid encapsulation, nanoparticle stability, and improved delivery efficiency. Additionally, phosphate buffers, Tris buffers, or citrate buffers are preferred to maintain appropriate pH. During storage and reconstitution, the pH range is preferably between 6.0 and 7.5, which suppresses degradation of nucleic acids and lipid components.

[0083] The lyophilized formulation of the present disclosure is characterized by the uniform dispersion of formulation components during the freeze-drying process and a particle size change of 80% to 120% after reconstitution. Reconstitution can be performed using methods commonly known in the pharmaceutical field, utilizing sterile water or suitable buffer solutions.(Freeze-Drying Process)

[0084] The conditions for freeze-drying suspensions of protein-bound nanoparticles containing nucleic acids are highly influential to maintaining the structural integrity of the protein-bound nanoparticles and preserving the activity of the nucleic acids. Freeze-drying of the present disclosure consists of freezing, primary drying, and secondary drying steps, each involving specific conditions to facilitate the stability of both the protein¬ bound nanoparticles and the nucleic acids.

[0085] The freezing step of the present disclosure is preferably carried out in the presence of 5 mM to 500 mM arginine. Rapid freezing at temperatures ranging from -40°C toNDJP.026WO PATENT-80°C is recommended, as it stabilizes the structure of protein-bound nanoparticles and reduces the risk of physical damage to the nucleic acids.

[0086] In the primary drying step, ice is sublimated under vacuum conditions. This process is typically conducted at a shelf temperature of -20°C to -10°C, with a pressure range of 50 to 200 mTorr. The drying time typically ranges from 10 to 48 hours, depending on the size of the protein-bound nanoparticles and the volume of the solution.

[0087] The secondary drying step removes residual moisture not eliminated during primary drying. In this step, the shelf temperature is typically increased to 20°C to 30°C, while maintaining a pressure of 50 to 200 mTorr. The drying time ranges from 2 to 24 hours, aiming for a final moisture content of 1% to 3%.

[0088] To confirm the quality of protein-bound nanoparticles post-freeze-drying, reconstitution tests may be performed. These include evaluating particle size uniformity, nucleic acid activity, and size distribution. If the reconstituted nanoparticles return to their original state, the freeze-drying conditions are deemed appropriate.

[0089] Thus, the pharmaceutical composition of the present disclosure facilitates the structural preservation of protein-bound nanoparticles and the stability of nucleic acids, enabling long-term storage even after freeze-drying.NDJP.026WO PATENT

EXAMPLES

[0090] The first solution was prepared by dissolving the lipid components listed in Table 1 in 100% ethanol at the ratios specified in Table 1 below. The total lipid concentration of this solution was adjusted so that the lipid concentration in the mixture obtained by combining the first and second solutions becomes 1.88 mM.Second Solution

[0091] The second solution was prepared using a solution containing 50 mM citrate and 263 mM sucrose (pH 3.5), ensuring a final nucleic acid concentration of 273 pg / mL. CleanCap mEGFP mRNA (5moll) (manufactured by TriLink BioTechnologies, Inc.) was used as the nucleic acid.Third Solution and Cryopreservation Solutions

[0092] The third solution or cryopreservation solutions included the following:Solution A: A solution containing 20 mM arginine and 263 mM sucrose (pH 7.4).Solution B: A solution containing 50 mM arginine and 263 mM sucrose (pH 7.4).Solution C: A solution containing 20 mM HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) and 263 mM sucrose (pH 7.4).NDJP.026WO PATENTSolution D: A 1× PBS solution prepared by diluting a 10× PBS solution (Gibco™10x Phosphate Buffered Saline) tenfold with ultrapure water, followed by dissolving sucrose to achieve a final concentration of 263 mM and adjusting the pH to 7.5.TE Buffer

[0093] The TE buffer was prepared by diluting TE Buffer (20×), RNase-free (manufactured by Invitrogen), twentyfold with ultrapure water and adjusting the pH to 7.5.Protein Solution

[0094] The protein solution was prepared using anti-CD8 antibodies and anti¬ human transferrin receptor antibodies.

[0095] Anti-CD8 antibody IgG: The heavy chain and light chain amino acid sequences correspond to Sequence ID Nos. 1 and 2, respectively. The heavy chain and light chain sequences of the Fab portion correspond to Sequence ID Nos. 3 and 4, respectively.

[0096] Anti-human transferrin receptor antibody IgG: The heavy chain and light chain amino acid sequences correspond to Sequence ID Nos. 5 and 6, respectively. The heavy chain and light chain sequences of the Fab portion correspond to Sequence ID Nos. 7 and 8, respectively. Cysteine residues were introduced into these IgGs and Fabs to enhance their ability to bind to the lipid nanoparticles (LNPs) via maleimide.

[0097] DNA sequences encoding these amino acid sequences were incorporated into CHO (Chinese Hamster Ovary cells) cells using mammalian expression vectors throughNDJP.026WO PATENTwell-known genetic engineering techniques to facilitate protein expression. The resulting IgGs and Fabs were treated with a reducing agent to undergo reduction, followed by dialysis to remove the reducing agent. The IgGs and Fabs were then dissolved in a solution containing 20 mM histidine acetate and 0.15 M NaCI (pH 5.5) to prepare a protein solution with a final protein concentration of 1-8 mg / mL.TABLE 1:Lipid Lipids Molar Ratio of Protein Composition Lipids(N / P)Composition 1 Compound1: Chol: DOPE: DMG-PEG(2K): 50:37.5:10:2:0.5 Anti-CD8 antibody DSPE-PEG(2K)-maleimide (6) IgG Composition 2 Compound1: Chol: DOPE: DMG-PEG(2K): 50:37.5:10:2:0.5 Anti-human transferrin DSPE-PEG(2K)-maleimide (6) antibody IgG Composition 3 Compound2: Chol: DSPC: DMG-PEG(2K): 50:37.5:10:1.5:1 Anti-CD8 antibody DSPE-PEG(2K)-maleimide (6) IgG Composition 4 Compound3: Chol: DOPE: DMG-PEG(2K): 50:37.5:10:2:0.5 Anti-CD8 antibody DSPE-PEG(2K)-maleimide (6) IgG Composition 5 Compound2: Chol: DOPE: DMG-PEG(2K): 50:37.5:10:2:0.5 Anti-CD8 antibody DSPE-PEG(2K)-maleimide (6) IgG Composition 6 Compound1: Chol: DOPE: DMG-PEG(2K): 50:37.5:10:2:0.5 Anti-CD8 antibody DSPE-PEG(2K)-maleimide (6) Fab Composition 7 Compound1: Chol: DOPE: DMG-PEG(2K): 50:37.5:10:2:0.5 Anti-human transferrin DSPE-PEG(2K)-maleimide antibody Fab(6)DOPE is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,DMG-PEG is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol,DSPE-PEG is N-[carbonyl-methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-NDJP.026WO PATENTphosphoethanolamine,DSPC is (1,2-distearoyl-sn-glycero-3-phosphocholine),Choi is cholesterol.DSPE-PEG(2K)-maleimide was obtained Avanti Research.Compounds 1-3

[0098] Compounds 1-3 in the present disclosure are lipids represented by the following chemical formulas:Compound 1:NDJP.026WO PATENTCompound 2:Compound 3:< MANUFACTURING CONDITIONS>[Particle Manufacturing Process]Manufacturing Condition 1-1:

[0099] Using the Automated Nanoparticle System (manufactured by Particle Works), the first solution was mixed at a flow rate of 2.25 mL / min with the second solution at a flow rate of 6.75 mL / min at 25°C to prepare a mixture. The resulting mixture was diluted with an equal volume of Solution A (used as the third solution). A protein solution was then added to the obtained mixture, and mixed. The molar ratio of the added protein to maleimide was adjusted to 0.1. The mixture containing the added protein solution was subjected to dialysisNDJP.026WO PATENTusing a Slide-a-Lyzer G3 dialysis membrane (10 kDa) (manufactured by Thermo Fisher) at 4°C for 20 hours. Solution A, used as the cryopreservation solution, was also employed as the dialysate. After dialysis, the solution inside the dialysis membrane was concentrated to approximately 1 mL using Amicon Ultra-15 100 kDa (manufactured by Merck Millipore), and the resulting solution was subjected to the separation process.Manufacturing Condition 1-2:

[0100] The same procedure as Manufacturing Condition 1-1 was conducted, except Solution B was used as the third solution and cryopreservation solution instead of Solution A.Manufacturing Condition 1-3:

[0101] The same procedure as Manufacturing Condition 1-1 was conducted, except Solution C was used as the third solution and cryopreservation solution instead of Solution A.Manufacturing Condition 1-4:

[0102] The same procedure as Manufacturing Condition 1-1 was conducted, except Solution D was used as the third solution and cryopreservation solution instead of Solution A.NDJP.026WO PATENT[Separation Process]Separating Condition 2-1:

[0103] To separate protein-bound lipid nanoparticles, Sepharose CL6B (manufactured by Cytiva) was used as the resin. A column packed with the resin was used with Econo-pac Chromatography Columns (manufactured by Bio-Rad). Solution A, used as the cryopreservation solution, served as the running buffer. After equilibrating the column packed with 15 mL of Sepharose CL6B by flushing it with Solution A for over an hour, a sample containing protein-bound lipid nanoparticles was added to the top of the column. The eluate from the bottom of the column was collected, and UV absorption at 280 nm was measured using Synergy2 (manufactured by Biotek) to identify fractions containing protein-bound lipid nanoparticles. The collected fractions were pooled and gently mixed by inversion to obtain a protein-bound lipid nanoparticle solution.Separating Condition 2-2:

[0104] The same procedure as Separating Condition 2-1 was conducted, except Solution B was used as the cryopreservation solution instead of Solution A.Separating Condition 2-3:

[0105] The same procedure as Separating Condition 2-1 was conducted, except Solution C was used as the cryopreservation solution instead of Solution A.NDJP.026WO PATENTSeparating Condition 2-4:

[0106] The same procedure as Separating Condition 2-1 was conducted, except Solution D was used as the cryopreservation solution instead of Solution A.Cryopreservation and Thawing

[0107] The protein-bound lipid nanoparticle solution obtained from the separation process was aliquoted into 5 mL glass vials and frozen at -80°C for 24 hours to obtain a cryopreserved formulation. The cryopreserved formulation was then incubated at 25°C for 1 hour to undergo the freeze-thaw process.< Analytical Conditions>N / P Ratio

[0108] The N / P ratio (nitrogen / phosphorus ratio) indicates the molar ratio of nitrogen atoms (N) in the lipid to phosphate groups (P) in the nucleic acid molecule and serves as an indicator for evaluating electrostatic interactions between nucleic acids and cationic components. Nitrogen atoms (N) refer to the total number of nitrogen atoms, such as those in amine groups, present in ionizable cationic lipids. Phosphate groups (P) refer to the total number of phosphate groups in the nucleic acid molecule. The count of nitrogen atoms was based on the number of cationic nitrogen atoms at pH 3.5. The N / P ratio was calculated by dividing the molar amount of nitrogen atoms in ionizable cationic lipids by the molar amount of phosphate groups in the nucleic acid.NDJP.026WO PATENTMeasurement of Z-Average Particle Size and PPI

[0109] The Z-average particle size and polydispersity index (PDI) of the protein-bound lipid nanoparticles were measured using a solution prepared by adding 20 pL of protein-bound lipid nanoparticle solution to 980 pL of 1 x PBS. Measurements were performed using a Zetasizer Nano ZS (manufactured by Malvern).

[0110] To evaluate the stability, the Z-average particle size was measured before freezing and after the freeze-thaw process. The percentage change in Z-average particle size was calculated, referred to as the particle change rate (%). A particle change rate between 80% and 120% was considered excellent, with 90% to 110% deemed particularly outstanding.Measurement of mRNA Encapsulation Efficiency

[0111] The mRNA encapsulation efficiency in protein-bound lipid nanoparticles was measured using the Ribogreen reagent (manufactured by Invitrogen).

[0112] The concentration of nucleic acids outside the protein-bound lipid nanoparticles was determined using a test solution (Solution X), which was prepared by diluting the nanoparticles in TE buffer without surfactants such as Triton-X. The total nucleic acid concentration, including nucleic acids inside and outside the nanoparticles, was determined using a test solution (Solution Y), prepared by diluting the nanoparticles in TE buffer containing 2.5% (w / w) Triton-X.

[0113] In a 96-well microplate, 100 pL of the Ribogreen reagent, diluted 200-fold in TE buffer, was mixed with 100 pL of Solution X or Solution Y. After incubating the mixture at 25°C for 5 minutes, the fluorescence intensity was measured with an excitation wavelengthNDJP.026WO PATENTof 485 nm and an emission wavelength of 528 nm. The nucleic acid concentration was calculated using a calibration curve.

[0114] The mRNA encapsulation efficiency (%) was calculated using the following formula:mB. NA Ejieaps’iiation Efficiency £%) ~Nucleic Acid iii Soh;tiCr'ii Y Nucleic Acid Coiiceiit iiori in tei-siirej e T Sointion X Nucleic Acid Gur’iceiitraiioii in Meksnrei u i SohitioTi YMeasurement of mRNA Recovery Rate

[0115] In the production of protein-bound lipid nanoparticles, the mRNA recovery rate was analyzed by comparing the amount of nucleic acid used during lipid nanoparticle formation with the amount of nucleic acid present in the final protein-bound lipid nanoparticle solution after the separation process. This analysis assessed the extent to which the input nucleic acid was successfully incorporated into the protein-bound lipid nanoparticles.

[0116] The mRNA recovery rate (%) was calculated using the following formula:mRNA Recovery Rate (%) =Amount of Nucleic Acids in the Protein-Bound Lipid Nanoparticle Solution after the Separation ProcessI \ - A; -mount of - Nucleic A:—etd -s in the S: -econd Solution - U -sed for Particl -e Formation / xIt’dNDJP.026WO PATENTAnalysis of Protein-Lipid Conjugates

[0117] A reducing solution was prepared by adding 5 mg of TCEP. HCI (manufactured by Thermo Fisher) to 100 pL of 1× PBS. The protein-bound lipid nanoparticle solution was diluted with 1× PBS to achieve an mRNA concentration of approximately 100 pg / mL, forming the sample. The reducing solution was mixed with the sample in equal volumes, and 1 / 3 volume of NuPAGE LDS Sample Buffer (4X) (manufactured by Invitrogen) was added, followed by thorough mixing.

[0118] The prepared loading sample was applied to Bolt Bis-Tris Plus Mini Protein Gels, 4-12% (manufactured by Invitrogen), and electrophoresis was performed at 100 V for 60 minutes using 1× MOPS / SDS running buffer. The electrophoresis apparatus used was the XCell SureLock Mini-Cell (manufactured by Invitrogen). The resulting gel was stained using the SilverQuest Silver Staining Kit (manufactured by Invitrogen) to confirm the formation of protein-lipid conjugates.< Results>

[0119] As shown in Table 2 below, Examples 1 to 8, which used solutions containing arginine as the third solution or cryopreservation solution, exhibited particle change rates for protein-bound lipid nanoparticles within the range of 80%-120%, indicating excellent stability against freeze-thaw processes. Notably, Examples 1 and 4 demonstrated particle change rates of 5% or less, reflecting exceptionally high stability against freeze-thaw processes.NDJP.026WO PATENT

[0120] In contrast, Comparative Examples 1 to 3, which did not use arginine, showed particle change rates of 814% for Comparative Example 1, 173% for Comparative Example 2, and 240% for Comparative Example 3. These results indicated that the particle size of protein-bound lipid nanoparticles significantly increased following freeze-thaw.

[0121] From these findings, it was confirmed that the use of arginine suppresses particle size changes caused by freeze-thaw operations and enhances particle stability.

[0122] Based on the above results, it was demonstrated that arginine effectively suppresses changes in the average particle size of protein-bound lipid nanoparticles during freeze-thaw processes.NDJP.026WO PATENT TABLE 2

[0123] The data for the protein-bound nanoparticles obtained with the following combinations are shown below:PDI Manufacturing Z-average Conjuga Z- Condition, particle mRNA (polydisp PDI Enca ted averageps ersity Separating size Anti bod particle before index) ulation Condition, and before size after y freezing Efficien after Lipid freezing Detectio freezecy (%) freezeComposition (nm) n thaw thaw1 - 12 - 1 84 0.058 93 YES 85 0.082 Example 1Composition 11 -22 -2 83 0.047 93 YES 93 0.309 Example 2Composition 1Comparative 1 -32 -3 91 0.133 91 YES 741 0.735 Example 1Composition 1Comparative 1 -42 -4 82 0.152 90 YES 142 0.120 Example 2Composition 1Comparative 1 -42 -4 99 0.141 90 YES 238 0.120 Example 3Composition 31 -42 -2 97 0.162 96 YES 101 0.160 Example 3Composition 31 - 12 - 1 94 0.056 94 YES 94 0.062 Example 4Composition 21 - 183 0.120 83 YES 86 0.173 Example 52 - 1NDJP.026WO PATENTManufacturing Z-average PD mR Conjuga Z- I Condition, particle NA (polydisp Separating size PDI Encaps ted averageAnti bod par ersity Condition, and before before ulation ticlesize after index) Lipid freezing freezing Efficien y after cy Detectio freezeComposition (%) n thaw freeze(nm) thawComposition 41 - 4Example 6 2 - 2 63 0.150 98 YES 65 0.126Composition 51 - 1Example 7 2 - 1 83 0.239 94 YES 78 0.190Composition 61 - 1Example 8 2 - 1 77 0.143 97 YES 73 0.143Composition 7

Claims

NDJP.026WO PATENTWHAT IS CLAIMED IS:

1. A pharmaceutical composition, comprising protein-bound nanoparticles and 5 mM to 500 mM of arginine.

2. The pharmaceutical composition according to claim 1, further comprising 10 mM to 2 M of a disaccharide.

3. The pharmaceutical composition according to claim 1 or 2, wherein the protein-bound nanoparticles comprise cationic lipids or ionizable cationic lipids, phospholipids, sterols, polyethylene glycol (PEG)-modified lipids, and conjugates of proteins and lipids.

4. A pharmaceutical composition, comprising protein-bound nanoparticles and arginine, wherein a concentration of arginine in the pharmaceutical composition before a lyophilization is 5 mM to 500 mM, wherein the pharmaceutical composition is in a form of a lyophilized formulation.

5. The pharmaceutical composition according to claim 4, further comprising a disaccharide, wherein a concentration of the disaccharide in the pharmaceutical composition before a lyophilization is 10 mM to 2 M.

6. The pharmaceutical composition according to claim 4 or 5, wherein the protein-bound nanoparticles comprise cationic lipids or ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and conjugates of proteins and lipids.

7. A method of manufacturing a pharmaceutical composition, comprising:NDJP.026WO PATENTfreezing protein-bound nanoparticles suspended in a freezing solution, wherein the freezing solution comprises 5 mM to 500 mM of arginine.

8. A method of manufacturing a protein-bound nanoparticle solution, comprising:mixing a nanoparticle solution and a protein solution to prepare a second mixed solution; andperforming a dialysis, a filtration, and / or a dilution of the second mixed solution using a freezing solution to obtain the protein-bound nanoparticle solution, wherein the freezing solution comprises 5 mM to 500 mM of arginine.

9. The method according to claim 8,wherein the nanoparticle solution is obtained by subjecting a first mixed solution comprised of a first solution and a second solution to a dialysis, a filtration, and / or a dilution using a third solution,wherein the first solution comprises ethanol and at least a lipid having a chemically reactive functional group in its molecule,the second solution is an aqueous solution comprising at least a drug, and the third solution contains 5 mM to 500 mM of arginine.

10. The method according to claim 8 or 9, further comprising freezing the protein-bound nanoparticle solution after the dialysis, the filtration, and / or the dilution.

11. The method according to any one of claims 8 to 10, wherein the freezing solution further comprises 10 mM to 2 M of a disaccharide.NDJP.026WO PATENT12. The method according to any one of claims 9 to 11, wherein the drug is a nucleic acid.

13. The method according to claim 9, wherein the chemically reactive functional group is a maieimide or an azide.

14. The method according to any one of claims 8 to 13, wherein:a pH of the freezing solution is 6.5 to 8.5,a pH of the second solution is 3.0 to 7.0, anda pH of the third solution is 6.5 to 8.5.

15. The method according to any one of claims 8 to 14, wherein the first solution further comprises cationic lipids or ionizable cationic lipids, sterols, phospholipids, and PEG-modified lipids.

16. The method according to any one of claims 8 to 15, wherein the arginine is selected from the group consisting of L-Arginine, L-Arginine Hydrochloride, L-Arginine Acetate, L-Arginine Phosphate, L-Arginine Sulfate, D-Arginine, a-Arginine, N-acetylarginine, Arginine Ethyl Ester, Arginine Sodium, Arginine Monolaurate, L-Arginine Pyroglutamate, Succinylated Arginine, N-Methylarginine, Dimethylarginine, Arginine a- Ketoglutarate, and combination thereof.

17. The method according to any one of claims 8 to 15, wherein the arginine is selected from the group consisting of L-Arginine, L-Arginine Hydrochloride, L-Arginine Acetate, L-Arginine Phosphate, L-Arginine Sulfate, and combination thereof.NDJP.026WO PATENT18. The method according to claim 11, wherein the disaccharide is selected from the group consisting of Sucrose, a-Trehalose, Leucrose, Murillosiose, Tricoccose, Melezitose, Thaumatinose, Lactosidol, Maltitol, Neotrehalose, Maltose, Lactose, Cellobiose, Trehalose, Isomaltose, Gentiobiose, Pleranose, Mannobiose, Laminaribiose, Sophorose, and combination thereof.

19. The method according to claim 11, wherein the disaccharide is Sucrose.-SO-

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