Protein-bound lipid nanoparticles and manufacturing method

Abstract

A protein-bound lipid nanoparticle, wherein an apparent pKa of the surface of the protein-bound lipid nanoparticle is 7.0 to 10.0, and the apparent pKa of the surface is measured by a TNS (p-toluidino-6-naphthalene sulfonic acid) measurement method. A method of manufacturing the protein-bound lipid nanoparticle, including: (a) mixing a first solution and a second solution to obtain a first mixed solution; (b) mixing the first mixed solution and a protein either simultaneously with step (a) or within an hour after completion of step (a) to obtain a second mixed solution; and (c) dialyzing, filtrating, or diluting the second mixed solution either simultaneously with step (b) or within an hour after completion of step (b), wherein the first solution includes ethanol and a lipid having a maleimide group in its molecule, the second solution includes a nucleic acid and a buffer, and the protein includes a thiol group.

Description

NDJP.025WO PATENTPROTEIN-BOUND LIPID NANOPARTICLES AND MANUFACTURING METHODINCORPORATION 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_025WO.xml created and last saved on March 13, 2026, which is approximately 5.7 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 protein-bound lipid nanoparticles and their manufacturing method.BACKGROUND

[0004] In the field of nucleic acid therapeutics, techniques to prevent rapid degradation of nucleic acids in the body are crucial. Additionally, nucleic acids face problems in cellular uptake in their native state. This creates a need for efficient methods to deliver nucleic acids into cells. One approach to address these problems is encapsulating nucleic acids within lipid nanoparticles. Lipid nanoparticles can preventNDJP.025WO PATENTnucleic acid degradation. They also utilize their properties to facilitate efficient cellular uptake of nucleic acids.

[0005] Recently, technologies for efficiently delivering nucleic acids to target cells have advanced. Concurrently, improvements in lipid nanoparticles are actively underway. For instance, methods are being developed to attach antibodies or ligands to the surface of lipid nanoparticles. These ligands or antibodies have high affinity for antigens expressed on target cells. PCT Publication No. WO2022 / 081694 discloses a technology that conjugates an anti-CD5 antibody to lipid nanoparticles for delivery to T cells. Similarly, "In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift", Biomaterials, 2021 Dec 29;281 :121339 discloses using an anti-CD3 antibody for the same purpose.

[0006] Two types of methods for attaching antibodies to the surface of lipid nanoparticles are known in the technical field relating to the present disclosure. One method (Method A) involves conjugating antibodies with maleimide-PEG lipids before forming lipid nanoparticles. Another method (Method B) forms lipid nanoparticles first, followed by antibody conjugation (See "Strategies for targeted gene delivery using lipid nanoparticles and cell-derived nanovesicles" Nanoscale Adv., 2023, 5, 3834-3856, and "In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift" Biomaterials, 2021 Dec29;281 : 121339). In the Method A, antibodies are treated with a reducing agent to generate free thiol groups. These thiol groups react with maleimide-PEG lipids. The resulting product is mixed with other lipids, such as ionizable cationic lipids and cholesterol. Lipid nanoparticles are then produced using techniques like ethanol injection. In the Method B, lipid nanoparticles are formed using lipids containing maleimide groups. The reduced antibodies are then conjugated with the lipid nanoparticles to produce antibody-conjugated lipid nanoparticles.NDJP.025WO PATENT

[0007] However, to the best of the applicant's knowledge, there is no disclosure in the prior art regarding protein-bound lipid nanoparticles in which an antibody is conjugated to lipid nanoparticles having a high pKa on its surface via a maleimide group in the context of Method B.SUMMARYProblems to be Solved by the Present Disclosure

[0008] The inventors of the present application conducted experiments to produce antibody-conjugated lipid nanoparticles. These experiments used lipids containing maleimide groups and antibodies with thiol groups. The results showed that the conventional method could produce antibody-conjugated lipid nanoparticles with many lipid compositions. However, it was difficult to manufacture such nanoparticles with certain lipid compositions.

[0009] Further investigation revealed key problems. When the apparent pKa of the surface of the lipid nanoparticles is high, the inventors discovered the conventional method faces inefficiencies. Antibody binding through maleimide groups does not proceed efficiently. Additionally, it becomes difficult to separate the antibody-conjugated lipid nanoparticles after conjugation.Means for Solving the Problems

[0010] In seeking to address the problems discussed above, the present disclosure provides manufacturing conditions that allow protein-binding through maleimide groups, even with lipid nanoparticles having a high pKa on its surface. The presentNDJP.025WO PATENTdisclosure also provides manufacturing conditions that enable efficient separation of protein-bound lipid nanoparticles after conjugation.

[0011] Moreover, the present disclosure provides protein-bound lipid nanoparticles compatible with lipid compositions with a high pKa on its surface. This technology facilitates the production of lipid compositions that were difficult to handle using conventional techniques.

[0012] Specifically, the present disclosure includes the following embodiments [1] to

[0021] :[1] A protein-bound lipid nanoparticle, wherein an apparent pKa of the surface of the protein-bound lipid nanoparticle is 7.0 to 10.0, and the apparent pKa of the surface is measured by a TNS (p-toluidino-6-naphthalene sulfonic acid) measurement method.[2] The protein-bound lipid nanoparticle according to [1] above, comprising a cationic lipid or an ionizable cationic lipid, a phospholipid, a sterol, a polyethylene glycol(PEG)-modified lipid, and a conjugate of protein and lipid, and encapsulating a nucleic acid.[3] A method of manufacturing the protein-bound lipid nanoparticle according to

[0001] or [2] above, comprising:(a) mixing a first solution and a second solution to obtain a first mixed solution;(b) mixing the first mixed solution and an unbound protein either simultaneously with said (a) or within an hour after completion of said (a) to obtain a second mixed solution; andNDJP.025WO PATENT(c) dialyzing, filtering, and / or diluting the second mixed solution either simultaneously with said (b) or within an hour after completion of said (b), wherein the first solution comprises ethanol and a lipid having a maleimide group in its molecule, said (b) being conducted under conditions selected to chemically bind the unbound protein to the lipid,the second solution comprises a nucleic acid and a buffer, and the unbound protein comprises a thiol group.[4] A method of manufacturing the protein-bound lipid nanoparticle according to

[0001] or [2] above, comprising:separating the protein-bound lipid nanoparticle and the unbound protein from a solution containing the protein-bound lipid nanoparticle and the unbound protein,wherein said separating comprises a gel filtration using a running buffer and a column packed with a resin, andthe resin is selected from the group consisting of a strong basic anion exchange resin and a weak basic anion exchange resin.[5] A method of manufacturing a protein-bound lipid nanoparticle, comprising:(a) mixing a first solution and a second solution to obtain a first mixed solution;(b) mixing the first mixed solution and an unbound protein either simultaneously with said (a) or within an hour after completion of said (a) to obtain a second mixed solution; and(c) dialyzing, filtering, and / or diluting the second mixed solution either simultaneously with said (b) or within an hour after completion of said (b),NDJP.025WO PATENTwherein the first solution comprises a lipid having a maleimide group in its molecule, said (b) being conducted under conditions selected to chemically bind the unbound protein to the lipid,the second solution comprises a nucleic acid,the unbound protein comprises a thiol group, andan apparent pKa of the surface of the protein-bound lipid nanoparticle is 7.0 to 10.0.[6] The method according to [5] above, further comprising:separating the protein-bound lipid nanoparticle and the unbound protein from a solution containing the protein-bound lipid nanoparticle and the unbound protein,wherein said separating comprises a gel filtration using a running buffer and a column packed with a resin,wherein the resin is selected from the group consisting of a strong basic anion exchange resin and a weak basic anion exchange resin.[7] The method according to [6] above, wherein the resin is DEAE (diethylaminoethyl cellulose) or Q-Sepharose.[8] The method according to any one of [5] to [7] above, wherein the first solution further comprises ethanol.[9] The method according to any one of [5] to [8] above, wherein the second solution further comprises a buffer.NDJP.025WO PATENT

[0010] The method according to any one of [5] to [9] above, wherein the apparent pKa of the surface of the protein-bound lipid nanoparticle is measured by a TNS (p-toluidino-6-naphthalene sulfonic acid) measurement method.

[0011] The method according to any one of [5] to

[0010] above, wherein the unbound protein is an antibody or an antigen-binding fragment thereof.

[0012] The method according to

[0011] above, wherein the antibody is an anti-CD8 antibody or an anti-human transferrin receptor antibody, wherein the anti-CD8 antibody comprises an amino acid sequence represented by Sequence ID No. 1 as a heavy chain and an amino acid sequence represented by Sequence ID No. 2 as a light chain, and the anti-human transferrin receptor antibody comprises an amino acid sequence represented by Sequence ID No. 3 as a heavy chain and an amino acid sequence represented by Sequence ID No. 4 as a light chain.

[0013] The method according to any one of [5] to

[0012] above, wherein the first mixed solution is mixed with the unbound protein within 30 minutes after the completion of said (a) to obtain the second mixed solution.

[0014] The method according to one of [5] to

[0013] above, wherein the apparent pKa of the surface of the protein-bound lipid nanoparticle is 9.0 to 10.0.

[0015] The method according to any one of [5] to

[0014] above, wherein the proteinbound lipid nanoparticle comprises a second lipid selected from the group consisting of a cationic lipid, an ionizable cationic lipid, a phospholipid, a sterol, and a polyethylene glycol(PEG)-modified lipid.NDJP.025WO PATENT

[0016] The method according to

[0015] above, wherein the cationic lipid is selected from the group consisting of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DC-Chol (3|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), CTAB (cetyltrimethylammonium bromide), and HEDC (2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan-aminium).

[0017] The method according to

[0015] above, wherein the ionizable cationic lipid is selected from the group consisting of DLin-MC3-DMA (1 ,2-dilinoleoyl-sn-glycero-3-methyl dimethylamine), DODAP (1,2-dioleoyl-3-dimethylaminopropane), DDA (dimethyldioctadecylammonium), C12-200 (1 ,1'-[[(2-(dimethylamino)ethyl)imino]bis-butanoyl]ethyl methylamine), OF-Deg-Lin (oxyfluoroalkyldeglinolein), ATLAS-75 (acylthiolipid amine 75), DLin-KC2-DMA (1 ,2-dilinoleoyl-sn-glycero-3-dimethylaminoethanol), DLin-DMA (1,2-dilinoleoyl-sn-glycero-3-dimethylamine), ALC-0315 dehydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyl-decanoate)), SM-102 ([(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(9-heptadecenoate)),and Compound 3:compound 3.

[0018] The method according to

[0015] above, wherein the phospholipid is selected from the group consisting of DSPC (1 ,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1 ,2-NDJP.025WO PATENTdipalmitoyl-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-phosphoglycerol), DSPG (1 ,2-distearoyl-sn-glycero-3-phosphoglycerol), DPPG (1 ,2-dipalmitoyl-sn-glycero-3-phosphoglycerol), 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-phosphoric acid), DPPA (1 ,2-dipalmitoyl-sn-glycero-3-phosphoric acid), and DSPA (1 ,2-distearoyl-sn-glycero-3-phosphoric acid).

[0019] The method according to

[0015] above, wherein the PEG-modified lipid is selected from the group consisting of mPEG-DSPE (methoxy-PEG-distearoylphosphatidylethanolamine), mPEG-DMG (methoxy-PEG-dimyristoylglycerol), mPEG-DOPE (methoxy-PEG-dioleoylphosphatidylethanolamine), mPEG-Ceramide (C14, C16, C18), mPEG-Cholesterol, and mPEG-DSG (methoxy-PEG-distearoylglycerol).

[0020] The method according to any one of [5] to

[0014] above, wherein the second mixed solution comprises a lipid mixture selected from the group consisting of: (i) Compound 1 , cholesterol, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-[carbonyl-methoxypolyethylene glycol]-1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), Compound 2, and DSPE-PEG-maleimide, and (ii) Compound 3, cholesterol, DOPE, N-[carbonyl-methoxypolyethylene glycol]- 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), Compound 2, and DSPE-PEG-maleimide,wherein said Compounds 1-3 are represented a chemical formula below:NDJP.025WO PATENTCompound 1 :OHCompound 2 :Compound3 :

[0021] A method of manufacturing a protein-bound lipid nanoparticle, comprising: separating a protein-bound lipid nanoparticle and an unbound protein from a solution containing the protein-bound lipid nanoparticle and the unbound protein, wherein said separating comprises a gel filtration using a running buffer and a column packed with a resin,NDJP.025WO PATENTwherein the resin is selected from the group consisting of a strong basic anion exchange resin and a weak basic anion exchange resin when an apparent pKa of the surface of the protein-bound lipid nanoparticle is the same as, within a range of ±0.2 of, or higher than a pH of the running buffer, andthe resin is selected from the group consisting of a strong acidic cation exchange resin and a weak acidic cation exchange resin when the apparent pKa of the surface of the protein-bound lipid nanoparticle is lower than the pH of the running buffer.Effects of the Present Disclosure

[0013] According to the present disclosure, even in the lipid nanoparticles having a high pKa on its surface, antibody conjugation via maleimide becomes feasible, and the separation conditions for lipid nanoparticles after antibody conjugation are improved. This enables the efficient production of antibody-conjugated lipid nanoparticles, even for lipid compositions that were conventionally difficult to handle.DETAILED DESCRIPTION(Protein-bound Lipid Nanoparticles)

[0014] In the present disclosure, the term "protein-bound lipid nanoparticle" refers to a structure in which at least one type of protein is chemically bound to lipid nanoparticles. These nanoparticles have a particle size ranging from 1 nm to 500 nm, preferably 10 nm to 200 nm, and more preferably 50 nm to 150 nm. This size range isNDJP.025WO PATENTsuitable for enhancing distribution to target tissues in vivo and intracellular uptake efficiency for applications such as drug delivery, diagnostics, and therapeutics.

[0015] The protein in the protein-bound lipid nanoparticles of the present disclosure includes, but is not limited to, enzymes, antibodies, antibody fragments, peptides, and their derivatives. These proteins are utilized to modify the surface of the particles, bind drugs, or impart functionality in vivo.

[0016] The proteins in the protein-bound lipid nanoparticles of the present disclosure may chemically bind to the nanoparticles through covalent chemical bonds, electrostatic interactions, hydrophobic interactions, and / or non-covalent adsorption. Among these, covalent chemical bonding is preferred due to its superior binding stability.

[0017] The protein-bound lipid nanoparticles of the present disclosure can be used as drug delivery carriers or vaccine adjuvants. In particular, lipid nanoparticles with antibodies or antibody fragments are useful for targeted therapies in cancer and infectious disease treatments. When used as a carrier for nucleic acid drug delivery, the lipid nanoparticles forming the protein-bound lipid nanoparticles are preferably composed of cationic lipids or ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and conjugates of proteins and lipids. This composition allows efficient incorporation of nucleic acids into the nanoparticles and effective delivery of nucleic acids to cells in vivo.

[0018] One embodiment of the protein-bound lipid nanoparticles in the present disclosure involves forming nanoparticles using a group of lipids comprising cationic lipids or ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and lipids with functional groups capable of chemical bonding, along with nucleic acids as drugs, and then chemically bonding antibodies to the particles via the functional groups capable of chemical bonding.NDJP.025WO PATENT

[0019] Another embodiment of the protein-bound lipid nanoparticles in the present disclosure involves forming nanoparticles using a group of lipids comprising cationic lipids or ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and conjugates of proteins and lipids, along with nucleic acids as drugs.(Surface pKa)

[0020] The method for measuring the apparent pKa of the surface (also referred to as “apparent surface pKa” hereinafter) of the protein-bound lipid nanoparticles or lipid nanoparticles is described below, utilizing a fluorescence probe method with TNS (6-p-toluidino-2-naphthalenesulfonic acid). This method takes advantage of the property that the fluorescence characteristics of TNS change according to its protonation state. First, a particle dispersion solution to be measured is prepared. Then, TNS is added at an appropriate concentration and uniformly dispersed in the particle dispersion solution. Subsequently, an acid or base is added to adjust the buffer solution, gradually varying the pH over a specific range to cover the target pH range. As a result, the protonation state of the particle surface changes, and the fluorescence intensity of TNS changes in a pH-dependent manner. The fluorescence intensity of TNS under each pH condition is measured using a fluorescence spectrophotometer, and the changes in fluorescence intensity are recorded. By analyzing the recorded fluorescence intensity data, the pH at which a sharp change in fluorescence intensity is observed is identified. This pH serves as an indicator of the apparent pKa of the surface of the lipid nanoparticles.

[0021] The apparent pKa of the surface of the protein-bound lipid nanoparticles can be measured under the conditions described by Jayaraman et al. "Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo" (Angewandte Chemie, Volume 124, Issue 34, pages 8657-8661, 2012).NDJP.025WO PATENT

[0022] For enhancing the functions of protein-bound lipid nanoparticles and lipid nanoparticles, an apparent pKa of the surface of the protein-bound lipid nanoparticles within the range of 6.0 to 9.6 is preferred. Within this range, the particles are expected to function effectively in vivo, particularly enhancing the delivery of nucleic acid drugs and uptake efficiency into target cells.

[0023] The inventors of the present application discovered a problem that when conjugating antibodies via maleimide, manufacturing becomes challenging when the apparent pKa of the surface of the protein-bound lipid nanoparticles is high. Furthermore, for lipid nanoparticles with a high apparent surface pKa, there is a possibility of decreased separation efficiency during the purification process of the protein-bound lipid nanoparticles after conjugation.

[0024] To address these issues, the manufacturing method of the present disclosure allows efficient antibody conjugation via maleimide even for lipid nanoparticles with an apparent surface pKa of 7.0-10.0, 7.5-10.0, 8.0-10.0, 8.5-10.0, 9.0-10.0, 9.5-10.0, or 9.0-9.5.

[0025] Furthermore, the manufacturing method of the present disclosure includes an improved separation step for recovering protein-bound lipid nanoparticles with high yield and high purity. For example, proper selection of the conditions of the resin used can enhance the recovery rate of the protein-bound lipid nanoparticles. Thus, the manufacturing method of the present disclosure is highly useful as it facilitates the production of protein-bound lipid nanoparticles with an apparent surface pKa of 7.0-10.0, which was conventionally difficult.

[0026] The term “apparent pKa” of the surface (also referred to as “apparent surface pKa”) of the protein-bound lipid nanoparticle in the present disclosure refers to anNDJP.025WO PATENTeffective acid dissociation constant of lipids and other surface-exposed functional groups in the nanoparticle environment. This apparent pKa may differ from the intrinsic pKa of the same functional groups in bulk solution due to the unique physicochemical properties of protein-bound lipid nanoparticle.(Particle Size)

[0027] The particle size (Z-ave) of lipid nanoparticles and protein-bound lipid nanoparticles in the present disclosure can be measured by dynamic light scattering (DLS) using a Malvern Zetasizer. To measure the Z-ave (Z-average particle size) using the Malvern Zetasizer, the particle suspension is appropriately diluted and injected into the measurement cell. The measurement is performed by selecting the Dynamic Light Scattering (DLS) mode, setting conditions such as temperature, viscosity, and refractive index, and initiating the measurement. The Brownian motion of the particles is analyzed from the scattered light intensity data to calculate the Z-ave. This method also provides the polydispersity index (PDI), which indicates the uniformity of the particle size distribution.(Cationic Lipids)

[0028] Cationic lipids used in the present disclosure are lipids carrying a positive charge. Examples of the cationic lipids of the present disclosure include DOTAP (1 ,2-dioleoyl-3-trimethylammonium-propane), DC-Chol (3p-[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),NDJP.025WO PATENTCTAB (cetyltrimethylammonium bromide), HEDC, a compound represented by the following chemical formula and others.Compound 1: 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan-aminium bromide (HEDC)

[0029] Cationic lipids generally exhibit cationic properties at a pH of 3.0 or higher, which results in lipid nanoparticles containing these lipids exhibiting high apparent surface pKa values. When the composition of the lipid nanoparticles includes a higher proportion of cationic lipids, the apparent surface pKa tends to increase further. The proportion of cationic lipids is preferably between 5 mol% and 50 mol% relative to the total lipids to appropriately adjust the apparent surface pKa of the lipid nanoparticles. Within this range, the lipid nanoparticles can maintain a desirable balance of characteristics without an excessively high apparent surface pKa. Particularly, a proportion of 10 mol% to 30 mol% is more preferable as it provides a favorable balance of particle stability, drug delivery efficiency, and interaction with cell membranes, making it highly suitable for pharmaceutical applications.NDJP.025WO PATENT(Ionizable Cationic Lipids)

[0030] Ionizable cationic lipids used in the present disclosure are lipids that can acquire a positive charge in a pH-dependent manner. They remain neutral at physiological pH and ionize in acidic environments. Examples of the ionizable cationic lipids of the present disclosure include DLin-MC3-DMA (1 ,2-dilinoleoyl-sn-glycero-3-methyl dimethylamine), DODAP (1 ,2-dioleoyl-3-dimethylaminopropane), DDA (dimethyldioctadecylammonium), C12-200 (1 ,1'-[[(2-(dimethylamino)ethyl)imino]bis-butanoyl]ethyl methylamine), OF-Deg-Lin (oxyfluoroalkyldeglinolein), ATLAS-75 (acylthiolipid amine 75), DLin-KC2-DMA (1 ,2-dilinoleoyl-sn-glycero-3-dimethylaminoethanol), DLin-DMA (1,2-dilinoleoyl-sn-glycero-3-dimethylamine), ALC-0315 ([(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyl-decanoate)), SM-102 ([(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(9-heptadecenoate)), and Compound 3.Compound 3

[0031] Ionizable cationic lipids or cationic lipids can be used individually or in combination with two or more types.NDJP.025WO PATENT(Phospholipids)

[0032] Phospholipids used in the present disclosure are amphiphilic lipid molecules composed of hydrophobic tails and hydrophilic heads containing a phosphate group and a polar group. Examples of the phospholipids of the present disclosure 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-phosphoglycerol), DSPG (1 ,2-distearoyl-sn-glycero-3-phosphoglycerol), DPPG (1 ,2-dipalmitoyl-sn-glycero-3-phosphoglycerol), 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-phosphoric acid), DPPA (1 ,2-dipalmitoyl-sn-glycero-3-phosphoric acid), and DSPA (1 ,2-distearoyl-sn-glycero-3-phosphoric acid). Phospholipids can be used individually or in combination of two or more types.(Sterols)

[0033] Sterols used in the present disclosure are lipid molecules with a steroid skeleton. Examples of the sterols of the present disclosure include cholesterol, epicholesterol, p-sitosterol, ergosterol, cholestanol, 7-dehydrocholesterol, androsterol, and desmethylcholesterol. Sterols can be used individually or in combination of two or more types.NDJP.025WO PATENT(PEG-Modified Lipids)

[0034] PEG-modified lipids used in the present disclosure are amphiphilic molecules with polyethylene glycol (PEG) chains attached to the lipid molecule ends. Examples of the PEG-modified lipids in the present disclosure include mPEG-DSPE (methoxy-PEG-distearoylphosphatidylethanolamine), mPEG-DMG (methoxy-PEG-dimyristoylglycerol), mPEG-DOPE (methoxy-PEG-dioleoylphosphatidylethanolamine), mPEG-Ceramide (C14, C16, C18), mPEG-Cholesterol, and mPEG-DSG (methoxy-PEG-distearoylglycerol). These lipids can be used individually or in combination of two or more types.(Lipids Containing Maleimide Groups)

[0035] The protein-bound lipid nanoparticles of the present disclosure are preferably prepared using lipids containing maleimide groups in their molecules. These lipids can be used to bind proteins. The hydrophobic regions of these lipid molecules can efficiently integrate into the lipid layer of the nanoparticles, while the maleimide groups are exposed on the nanoparticle surface, allowing chemical bonding with unbound proteins. As a result, proteins can be immobilized on the nanoparticles. Using lipids with maleimide groups facilitates functionalization and stabilization of the nanoparticle surface. Maleimide groups effectively react with thiol groups, enabling protein-binding. For example, by introducing maleimide groups into the lipid molecules on the nanoparticle surface, stable thioether bonds can be formed by reaction of such maleimide groups with the thiol groups of unbound proteins.

[0036] Maleimide-PEG-DSPE (manufactured by Avanti Research) is an example of a lipid containing maleimide groups, which enhances nanoparticle stability and biocompatibility through the PEG chains. By incorporating such lipids into the lipidNDJP.025WO PATENTnanoparticles, efficient binding of proteins or pharmaceutical components is achieved. For instance, maleimide-PEG-DSPE can be arranged on the nanoparticle surface, where it reacts with the thiol groups of unbound proteins to construct protein-bound lipid nanoparticles. This process can be performed under mild conditions, minimizing structural changes in the proteins while achieving high binding efficiency.

[0037] However, the inventors of the present disclosure discovered a problem that the protein-binding ability of maleimide significantly decreases when used in conjunction with lipids containing quaternary ammonium groups, such as HEDC. The mechanism of this phenomenon is not fully understood; however, the inventors consider that the quaternary ammonium ion may act as a Lewis acid, promoting the hydrolysis of the maleimide group, thereby reducing the protein-binding ability of the maleimide.

[0038] To address this issue, the manufacturing method of the present disclosure facilitates rapid contact between maleimide groups and thiol groups in the presence of quaternary ammonium compounds. Moreover, the inventors of the present application also discovered a problem that the presence of ethanol during the reaction between maleimide and unbound proteins significantly reduces binding efficiency. To overcome this problem, the manufacturing method of the present disclosure includes a step to promptly remove ethanol either simultaneously with or after the reaction between maleimide and proteins through dialysis, filtration, or dilution.

[0039] The inventors of the present disclosure have identified that such manufacturing improvements are helpful for enhancing the binding of maleimide and proteins in the presence of compounds with quaternary ammonium groups.NDJP.025WO PATENT(Conjugates of Protein and Lipid)

[0040] In one embodiment of the present disclosure, the conjugates of protein and lipid have a structure in which lipid molecules are chemically bound to protein molecules. These conjugates contribute to surface modification of nanoparticles, imparting target specificity and enhancing biocompatibility. The binding between proteins and lipids may be achieved by introducing functional groups capable of covalent chemical bonding into the lipid molecules. For example, a stable thioether bond can be formed by reacting maleimide groups in the lipid molecules with thiol groups in the unbound protein molecules. This method has the advantage of proceeding under mild conditions, minimizing the impact on protein structure and activity.

[0041] As lipid molecules, compounds such as phospholipids with hydrophobic chains (e.g., DSPE or DPPC) combined with polyethylene glycol (PEG) are preferable, as they facilitate the binding or immobilization of proteins on the surface of lipid nanoparticles. Proteins include antibodies, antigen-binding fragments, enzymes, peptides, or derivatives thereof.

[0042] A specific example is the conjugate obtained by reacting maleimide-PEG-DSPE with thiol groups of antibody molecules. This conjugate can be used to immobilize antibodies on the surface of lipid nanoparticles, exhibiting high binding affinity to specific cells or target molecules.(Nucleic Acids)

[0043] Nucleic acids of the present disclosure include mRNA, siRNA, shRNA, DNA, antisense oligonucleotides, and plasmid DNA, which are utilized for regulating gene expression or suppressing the expression of target genes.NDJP.025WO PATENT(mRNA)

[0044] mRNA of the present disclosure is an RNA molecule that carries genetic information for protein synthesis and serves as a template within cells. It transports information transcribed from genes to ribosomes to facilitate the synthesis of specific proteins. The structure of mRNA may include a cap structure, a 5' untranslated region (5' UTR), a coding region, a 3' untranslated region (3' UTR), and a poly-A tail, but it is not limited to these elements. To reduce the immunogenicity of mRNA and enhance its stability and translation efficiency, modifications such as substituting uridine with 1-methyl-3'-pseudouridine or 5-methylcytidine may be introduced.(siRNA)

[0045] siRNA of the present disclosure is an RNA molecule with gene-silencing functionality that suppresses the expression of specific genes. siRNA typically consists of double-stranded RNA (dsRNA) of 20-25 nucleotides, with annealed sense and antisense strands featuring 5' phosphate groups, although it is not limited to this structure. To improve the stability and reduce the immunogenicity of siRNA, as well as to enhance specificity for target mRNA, chemical modifications such as 2'-methoxyuridine, 2'-fluoro modifications, or phosphorothioate linkages can be introduced.(miRNA)

[0046] miRNA of the present disclosure is a small non-coding RNA molecule that functions to suppress the expression of specific genes within cells. miRNA is typically about 20-22 nucleotides in length and regulates protein expression by binding complementarily to target mRNA, thereby suppressing gene expression or post-transcriptionally modulating it. To enhance the stability of miRNA and its binding efficiencyNDJP.025WO PATENTto target mRNA, chemical modifications such as 2'-0-methylation or the introduction of phosphorothioate linkages may be employed.(Manufacturing Method)

[0047] The manufacturing method of the protein-bound lipid nanoparticles in the present disclosure preferably includes a lipid nanoparticle preparation step, a protein mixing step, a substitution step, and a separation step. These steps are preferably carried out in the order of lipid nanoparticle preparation, protein mixing, substitution, and separation.

[0048] In the lipid nanoparticle preparation step, a first solution and a second solution are mixed to obtain a first mixed solution. The first solution contains at least ethanol and a lipid having a maleimide group, while the second solution contains nucleic acids and a buffer. The resulting first mixed solution is a solution containing lipid nanoparticles encapsulating nucleic acids. These lipid nanoparticles include maleimide groups, which lose their protein-binding ability rapidly in the presence of compounds containing quaternary ammonium groups.

[0049] Therefore, the manufacturing method of the present disclosure includes a protein mixing step in which the mixing of the first mixed solution with proteins having thiol groups is initiated either simultaneously with or within one hour after the lipid nanoparticle preparation step, resulting in a second mixed solution. This ensures early contact between maleimide groups and proteins, facilitating efficient bonding regardless of the presence of quaternary ammonium compounds.

[0050] The first mixed solution contains ethanol derived from the first solution. Since ethanol can reduce the efficiency of chemical bonding between maleimide andNDJP.025WO PATENTproteins, its early removal is also highly preferable. Accordingly, the manufacturing method of the present disclosure includes a substitution step in which dialysis, filtration, and / or dilution of the second mixed solution to remove ethanol is initiated simultaneously with or within one hour after the protein mixing step.

[0051] The separation step in the present disclosure is a step of separating protein-bound lipid nanoparticles from unbound proteins using a solution containing protein-bound lipid nanoparticles and unbound proteins. The solution containing proteinbound lipid nanoparticles and unbound proteins can be obtained, for example, through the lipid nanoparticle preparation step, protein mixing step, and substitution step of the present disclosure, but is not limited thereto.

[0052] In the present disclosure, a concentration step may optionally be performed to increase the concentration of protein-bound lipid nanoparticles to a high level, either before or after the separation step. This step can be carried out using known methods, such as concentration with ultrafiltration membranes.(Lipid Nanoparticle Preparation Step)

[0053] In the lipid nanoparticle preparation step of the present disclosure, a first solution and a second solution are mixed to obtain a first mixed solution.(First Solution)

[0054] The first solution in the present disclosure contains at least ethanol and a lipid having a maleimide group. The first solution may optionally include lipids such as cationic lipids, ionizable cationic lipids, phospholipids, cholesterol, or PEG-modified lipids. The first solution is a solution in which these lipids are dissolved in ethanol.NDJP.025WO PATENT(Second Solution)

[0055] In the present disclosure, the second solution comprises a nucleic acid and a buffer.

[0056] The second solution may optionally include a buffer and a metal chloride. It is a solution in which the nucleic acid drug is dissolved in water. The pH of the second solution is preferably in the range of 3.0 to 7.0, with 3.0 to 5.0 being particularly preferred. The concentration of the buffer in the second solution is preferably 10 to 200 mM, and more preferably 10 to 100 mM.

[0057] Under these conditions, the efficiency of the chemical bonding between the unbound protein and the maleimide group on the surface of the lipid nanoparticles is further enhanced.(First Mixed Solution)

[0058] The first mixed solution in the present disclosure is a solution obtained by mixing the first solution and the second solution and contains lipid nanoparticles encapsulating nucleic acids.(Mixing)

[0059] The mixing of the first solution and the second solution is not particularly limited and can be performed using various methods. For example, the first and second solutions can be supplied from independent sources simultaneously and mixed directly in a mixing device, or the first solution can be introduced into a mixing vessel first, followed by the addition of the second solution. Desired mixing characteristics can be achieved by controlling the mixing speed, mixing time, and mixing temperature during the process.NDJP.025WO PATENTAdditionally, a stirring device can be used for mixing the first and second solutions, with adjustments to the shape of the stirring blade, rotational speed, and rotation direction as needed. Preheating or cooling at least one of the solutions before mixing may also improve the physical properties after mixing. Furthermore, ultrasonic treatment, pressurization, or depressurization can be applied during the mixing process to enhance uniformity and reaction efficiency. A flow channel design where the first and second solutions are supplied via separate routes and merged just before the mixing point can improve process continuity. The mixing methods of the present disclosure are not limited to any single approach and can combine multiple methods as appropriate. Through such mixing, the first mixed solution, formed by combining the first and second solutions, is obtained.(Protein Mixing Step)

[0060] In the protein mixing step of the present disclosure, the mixing of the first mixed solution and the unbound protein begins either simultaneously with or within one hour after the lipid nanoparticle preparation step. “Simultaneously with the lipid nanoparticle preparation step” means starting the mixing of the first mixed solution and the unbound protein during the preparation of the first mixed solution. If mixing begins within one hour after the lipid nanoparticle preparation step, the maleimide remains capable of chemical bonding with the unbound protein even in the presence of compounds with quaternary ammonium groups. Starting the mixing process within one hour, and preferably within 30 minutes or more preferably within 15 minutes, further enhances the efficiency of chemical bonding.NDJP.025WO PATENT(Protein)

[0061] In the protein mixing step, unbound proteins having thiol groups are used. The proteins are not particularly limited as long as they include thiol groups, but aqueous solutions are preferred over powdered forms. In aqueous solutions, a pH range of 5.0 to 8.0 is preferred, with a pH range of 5.0 to 6.0 being particularly advantageous, as it helps to suppress the formation of disulfide bonds by free thiol groups. Examples of aqueous protein solutions include antibody solutions, antibody fragment solutions, enzyme solutions, peptide solutions, or ligand solutions, which can be used individually or in combination. The thiol groups form conjugates with the maleimide groups present in the lipid nanoparticles in the first mixed solution. When mixing begins, the unbound proteins react with the maleimide groups and bind to the lipid nanoparticles. The reaction between the unbound proteins and lipid nanoparticles is preferably conducted gently at a pH of 6.5-7.5 over several hours to overnight.(Substitution Step)

[0062] The substitution step in the present disclosure starts dialysis, filtration, and / or dilution of the second mixed solution either simultaneously with or within one hour after the protein mixing step. This step is essential to promptly remove or dilute ethanol or other substances from the immediate vicinity of the reaction site between the unbound protein and the maleimide group on the lipid nanoparticles. “Simultaneously with the protein mixing step” means beginning the dialysis, filtration, and / or dilution of the second mixed solution during the mixing of the first mixed solution and the unbound protein. Starting the substitution process within one hour of completing the protein mixing step avoids significant reductions in the chemical bonding efficiency of the maleimide with theNDJP.025WO PATENTunbound protein. Preferably, the substitution process begins within 30 minutes, and more preferably within 15 minutes, to achieve desired results.(Dialysis, Filtration, and / or Dilution)

[0063] Dialysis of the present disclosure is performed to remove ethanol, unencapsulated nucleic acids, and unnecessary low-molecular-weight components from the second mixed solution, using a dialysis bag or dialysis cartridge. The molecular weight cutoff (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 inside the dialysis membrane. When retaining lipid nanoparticles or proteinbound lipid nanoparticles inside the dialysis membrane, it is preferable to use a dialysis membrane with a molecular weight cutoff (MWCO) of 10,000-100,000 Da and perform dialysis at 4°C or room temperature for approximately 12-48 hours.

[0064] Filtration of the present disclosure can be performed by repeated cycles of concentration and dilution using an ultrafiltration membrane, allowing the removal of ethanol, unencapsulated nucleic acids, and unwanted low-molecular-weight components.(Separation Step)

[0065] In one embodiment of the present disclosure, the manufacturing method includes a separation step to separate protein-bound lipid nanoparticles from unbound proteins using a solution containing both. The separation process involves gel filtration using a running buffer and a resin-filled column. The resin is selected from strong and weak basic anion exchange resins when the apparent surface pKa of the proteinbound lipid nanoparticles is equal to or higher than the pH of the running buffer. ForNDJP.025WO PATENTnanoparticles with an apparent surface pKa lower than the running buffer pH, the resin is selected from strong and weak acidic cation exchange resins.(Method for Producing Protein-Bound Lipid Nanoparticles According to One Embodiment of the Present Disclosure)

[0066] The method for producing protein-bound lipid nanoparticles according to one embodiment of the present disclosure includes a separation step of separating protein-bound lipid nanoparticles from unbound proteins using a solution containing protein-bound lipid nanoparticles and unbound proteins.

[0067] The separation step includes a gel filtration process using a column packed with resin and a running buffer. The resin is selected from the group consisting of strongly basic anion exchange resins and weakly basic anion exchange resins.

[0068] The protein-bound lipid nanoparticles include cationic lipids or ionizable cationic lipids, phospholipids, sterols, PEG-modified lipids, and conjugates of proteins and lipids. They encapsulate nucleic acids, have an apparent surface pKa of 7.0 to 10.0, and the apparent surface pKa is measured using the TNS measurement method.

[0069] The method for producing protein-bound lipid nanoparticles according to one embodiment of the present disclosure comprises:(a) Lipid Nanoparticle Preparation Step:Mixing a first solution with a second solution to obtain a first mixture. The first solution comprises ethanol and a lipid having a maleimide group in its molecule. The second solution comprises a nucleic acid and a buffer. (b) Protein Mixing Step:Initiating mixing of the first mixture and an unbound protein to obtain aNDJP.025WO PATENTsecond mixture, either simultaneously with or within one hour after completing the lipid nanoparticle preparation step. The mixing is conducted under conditions selected to chemically bind the unbound protein to the lipid via reaction with the maleimide group.(c) Substitution Step:Initiating dialysis, filtration, and / or dilution of the second mixture either simultaneously with or within one hour after completing the protein mixing step.(d) Separation Step:Separating protein-bound lipid nanoparticles from unbound proteins using the solution obtained from the substitution step, which contains proteinbound lipid nanoparticles and unbound proteins. The separation step includes a gel filtration process using a column packed with a resin and a running buffer.

[0070] The first solution contains at least a lipid having a maleimide group in its structure and ethanol. The second solution contains nucleic acids and a buffer. The unbound protein contains thiol groups capable of reacting with the maleimide groups.Selection of Resins:

[0071] When the apparent surface pKa of the protein-bound lipid nanoparticles is equal to or higher than the pH of the running buffer, the resin is selected from the group consisting of strongly basic anion exchange resins and weakly basic anion exchange resins.NDJP.025WO PATENT

[0072] When the apparent surface pKa of the protein-bound lipid nanoparticles is lower than the pH of the running buffer, the resin is selected from the group consisting of strongly acidic cation exchange resins and weakly acidic cation exchange resins.(Gel Filtration Process)

[0073] In the present disclosure, the separation step includes a gel filtration process using a running buffer and a column filled with resin. The gel filtration process is a method for separating or purifying components in a solution, particularly employing gel filtration technology based on molecular size. Gel filtration is a chromatography technique that separates molecules or complexes in solution based on their size. By using porous carrier materials, molecules or complexes are separated depending on their ability to enter and exit the pores of the resin. Larger components that cannot enter the pores pass through the column more quickly, while smaller molecules that traverse the pores are delayed. If the resin exhibits affinity for certain components in the solution, the passage speed can be significantly affected. Thus, separation occurs based on the molecular size and properties of the components in the solution.

[0074] The present disclosure aims to improve the separation efficiency and reproducibility of gel filtration, particularly for the separation of high-molecular-weight substances or the removal of low-molecular-weight compounds. The resin used in the present disclosure has an average particle size of 10-300 pm and pore diameters ranging from 2-300 nm. The carrier material may be chemically modified agarose or polyacrylamide. The gel filtration column operates at a flow rate of 0.1-5 mL / min, a temperature range of 4-50°C, and a buffer pH of 3-10.

[0075] Generally, in protein purification techniques using ion-exchange resins, it is well known to bind proteins to the resin by utilizing charge while delivering a runningNDJP.025WO PATENTbuffer and then elute the proteins from the resin using an elution solution with a composition different from that of the running buffer.

[0076] In the gel filtration step of the present disclosure, ion-exchange resins may be used; however, the gel filtration step of the present disclosure preferably does not adopt a method of eluting proteins using an elution solution. Instead, it employs a method of separating protein-bound lipid nanoparticles over time while delivering the running buffer. This approach enables separation in a short period of time. Generally, elution solutions often have high salt concentrations or pH values outside the neutral range. By avoiding the combination of elution solutions and performing separation using only the flow of the running buffer, chemical stress on the protein-bound lipid nanoparticles can be reduced, allowing purification of the protein-bound lipid nanoparticles under mild conditions.

[0077] In the gel filtration step of the present disclosure, gel filtration may be carried out using a combination of resins with ion-exchange functionality and resins without ion-exchange functionality. Alternatively, multiple resins with ion-exchange functionality may be combined and used.(Running Buffer)

[0078] In the gel filtration process using a solution containing protein-bound lipid nanoparticles and unbound proteins, the running buffer is supplied to the resin-filled column. As the protein-bound lipid nanoparticles pass through the resin, the unbound proteins are separated. Suitable running buffers include phosphate, citrate, or HEPES as buffering agents, as these maintain stable pH and minimize damage to the bioactivity of the protein-lipid nanoparticles. The pH of the buffer is preferably set between 6.5 and 8.5,NDJP.025WO PATENTwith a more preferable range of 7.0 to 7.5. The buffer concentration is preferably 0.5 mM to 100 mM.

[0079] Metal chlorides may be added, typically 50 mM to 200 mM of sodium chloride or potassium chloride, to prevent aggregation of the protein-bound lipid nanoparticles and to enhance separation efficiency.

[0080] Disaccharides may be added, typically 50 mM to 500 mM of sucrose, to prevent aggregation of the protein-bound lipid nanoparticles.(Resins Used in the Column)

[0081] In the present disclosure, the resin is selected from the group consisting of strong basic anion-exchange resins and weak basic anion-exchange resins when the apparent surface pKa of the protein-bound lipid nanoparticles is higher than the pH of the running buffer. Furthermore, when the apparent surface pKa of the protein-bound lipid nanoparticles is lower than the pH of the running buffer, the resin is selected from the group consisting of strong acidic cation-exchange resins and weak acidic cation-exchange resins. The resin in the present disclosure may also be combined with specialized chemical functional group anion-exchange resins, macroporous anion-exchange resins, and customized resins.

[0082] Specific examples of strong basic anion-exchange resins include Amberlite IRA-400, Amberlite IRA-900, Amberlite IRA-910, Amberlite IRA-96, Amberlite FPA98CI, Dowex 1, Dowex21K, Dowex Monosphere 550A, Dowex Monosphere 650C, Q Sepharose FF (Fast Flow), Q Sepharose XL, Q Sepharose Big Beads, Q Sepharose High Performance, AG 1-X8, AG 1-X4, Capto Q, Macro-Prep Q, Tulsion A-23, Lewatit MonoPlus M 500, Purolite A500, Purolite A520E, Purolite A860, Diaion SA10A, LanxessNDJP.025WO PATENTSR-7, Diaion SA20A, ResinTech SBG1 , ResinTech SBG2, Amberlite FPA53, Amberjet 4400, Amberjet 4500, and Tulsion T-52.

[0083] Examples of weak basic anion-exchange resins include Amberlite IRA-67, Amberlite IRA-93, Amberlite IRA-45, Amberlite FPA51 , Dowex 2, Dowex MWA-1 , DEAE Sepharose Fast Flow, DEAE Sepharose CL-6B, DEAE Cellulose, DEAE-Trisacryl M, DEAE-Fractogel EMD, Macro-Prep DEAE, Capto DEAE, QAE Sephadex A-25, QAE Sephadex A-50, Bio-Rex 9, Diaion WA30, Diaion WA20, Tulsion A-32, Lewatit MP 62, Lewatit MP 64, Amberjet 1200, Amberjet 1500, ResinTech SBA1, Amberlite FPA96CI, Sepharose DEAE FF, and Tosoh TSKgel DEAE-5PW. Examples of chelating anion-exchange resins include Chelex 100, Amberlite IRC-748, Amberlite IRC-50, Dowex M4195, Dowex A-1, Duolite C-467, Lewatit TP 207, Purolite S930, Purolite S940, Chelating Sepharose Fast Flow, Diaion CR11 , Diaion CR20, Purolite S910, Purolite S950, Tulsion CH-90, Lanxess TP 260, Mitsubishi RCP142, Lewatit TP 214, Nippon RCP110, Amberlite IRC-747, ResinTech MTS9500, ResinTech ASM1 OHP, Macro-Prep IMAC, and IMAC Sepharose 6FF. Examples of resins with specialized functional groups include Phenyl Sepharose, Pyridyl Sepharose, Capto Adhere, Blue Sepharose, Red Sepharose, Green A Sepharose, Heparin Sepharose, Butyl Sepharose, Octyl Sepharose, Hydrophobic Interaction Resins, Trisacryl Q, Poly-DMAA Resins, Affinity Sorbents AS1 , Affinity Sorbents AS2, Immobilized Metal Affinity Chromatography (IMAC) Resins, Hydroxyapatite Resins, Sepharose XL, Amberlite XAD7HP, Amberlite XAD16, Lewatit K2620, and Lanxess K2020. Examples of macroporous anion-exchange resins include Amberlite IRA-910, Amberlite IRA-458, Dowex Optipore L-493, Dowex Monosphere 77, Dowex Monosphere 78, Purolite A600, Purolite A830, Purolite A300, Tulsion A-45, Diaion SA20A, Lewatit MP 500, Lewatit MP 600, Lanxess MP 64, Mitsubishi MP32, Nippon RMP110, Macro-Prep High Q, Macro-Prep High S, Amberlite IRA-68, Amberlite FPA111 ,NDJP.025WO PATENTand Amberlite FPX66. Other specialized resins include AG 1-X4, AG 2-X8, AG MP-1, QAE-Sephadex A-25, Mono Q, Resource Q, Tosoh TSKgel QAE-5PW, Bio-Gel P-2, Superdex75, Superdex200, ResinTech SIR-100, ResinTech SIR-200, Lanxess lonacSR-7, Diaion PK212, Amberlite FPC3500, Lewatit AP246, Tosoh TSKgel CM-5PW, and Sephadex LH-20.

[0084] When the apparent surface pKa of protein-bound lipid nanoparticles is the same as, within a range of ±0.2 of, or higher than the pH of the running buffer, a significant proportion of the protein-bound lipid nanoparticles become positively charged in the running buffer. Conversely, when the apparent surface pKa is lower than the pH of the running buffer, the protein-bound nanoparticles may become negatively charged. This relationship allows for the selection of resins based on the apparent surface pKa of the protein-bound nanoparticles and the pH of the running buffer.

[0085] In one embodiment of the present disclosure, when the apparent surface pKa of the protein-bound lipid nanoparticles is in the range of 7.0 to 10.0 and the running buffer pH is 6.0 to 7.0, the resin is selected from the group consisting of strong basic anion-exchange resins and weak basic anion-exchange resins. The separation efficiency of the protein-bound lipid nanoparticles is further improved when the resin is DEAE (diethylaminoethyl cellulose) or Q-Sepharose. Q-Sepharose provides particularly enhanced efficiency.NDJP.025WO PATENT[EXAMPLES]<Materials>First Solution

[0086] 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 to produce the compositions 1-3 respectively. The total lipid concentration of the first solution was adjusted so that the lipid concentration in the mixture obtained by combining the first and second solutions was 1.88 mM.Second Solution

[0087] The second solution was prepared using a 50 mM citrate buffer (pH 3.5) (manufactured by Spectrum Chemicals & Lab Products) to achieve a final nucleic acid concentration of 273 pg / mL. CleanCap mEGFP mRNA (5moU) (manufactured by TriLink BioTechnologies, Inc.) was used as the nucleic acid.Third Solution

[0088] The third solution was a mixture containing 20 mM HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid) and 263 mM sucrose, adjusted to pH 7.4.lx PBS Solution

[0089] The 1* PBS solution was prepared by diluting a 10* PBS solution (Gibco™ 10* Phosphate Buffered Saline) tenfold with ultrapure water and adjusting the pH to 7.5.TE BufferNDJP.025WO PATENT

[0090] The TE buffer was prepared by diluting a 20* TE Buffer (RNase-free) (manufactured by Invitrogen) twentyfold with ultrapure water and adjusting the pH to 7.5.Protein Solution

[0091] The protein solution was made with anti-CD8 antibodies and antihuman transferrin receptor antibodies. The anti-CD8 IgG was derived from heavy and light chains with amino acid sequences shown as Sequence ID No. 1 and Sequence ID No. 2, respectively. The anti-human transferrin receptor IgG had sequences corresponding to Sequence ID No. 3 for the heavy chain and Sequence ID No. 4 for the light chain.

[0092] Cysteine residues were introduced into these IgGs to enhance their ability to bind to lipid nanoparticles (LNPs) via maleimide. Using well-known genetic engineering techniques, DNA sequences encoding these amino acid sequences (Sequence ID No. 1-4) were incorporated into CHO cells (Chinese Hamster Ovary cells) via mammalian cell expression vectors to facilitate protein expression. The resulting IgGs were treated with a reducing agent to undergo reduction, followed by dialysis to remove the reducing agent. The IgGs 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 1Lipid Composition Lipids Molar Ratio of Lipids Protein (N / P)Composition 1 Compoundl :Chol:DOPE:DSPE- 30:33:30:3:3.5:0.5 (3.5) Anti-CD8 antibody PEG(2K):Compound2:DSPE-PEG(2K)- IgGmaleimideComposition 2 Compoundl :Chol:DOPE:DSPE- 30:33:30:3:3.5:0.5 (3.5) Anti-human PEG(2K):Compound2:DSPE-PEG(2K)- transferrin antibodyNDJP.025WO PATENTLipid Composition Lipids Molar Ratio of Lipids Protein (N / P)maleimide igG Composition 3 Compounds: 45:23:25:3:3.5:0.5 (4.5) Anti-human Chol:DOPE:DPPE- transferrin antibody PEG(2K):Compound2:DSPE-PEG(2K)- IgGmaleimideDOPE is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,DSPE-PEG is N-[carbonyl-methoxypolyethylene glycol]- 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,DPPE-PEG is N-[carbonyl-methoxypolyethylene glycol]-1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine,Choi is cholesterol.DSPE-PEG(2K)-maleimide was obtained from Avanti Research.Compounds 1-3

[0093] Compounds 1-3 in the present disclosure are lipids represented by the following chemical formulas.Compound 1 : 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan-aminium bromide (HEDC)ooNDJP.025WO PATENTCompound 2 :Compound 3 :Manufacturing Conditions>(Particle Manufacturing Process)[Manufacturing Condition 1-1]

[0094] Using the Automated Nanoparticle System (manufactured by Particle Works), the first solution was mixed at a flow rate of 2.25 mL / min and the second solution was mixed at a flow rate of 6.75 mL / min at 25°C to prepare the first mixture. The obtained first mixture was diluted with an equal volume of the third solution to obtain a diluted first mixture. Thirty minutes after obtaining the first mixture, a protein solution was added and mixed with the diluted first mixture to obtain a second mixture. The molar ratio of the added protein to maleimide was adjusted to 0.1. Thirty minutes after the addition of the protein solution, dialysis of the second mixture containing the added protein solution was initiated.NDJP.025WO PATENTThe dialysis was conducted at 4°C for 16 hours using a Slide-a-Lyzer G3 dialysis membrane (10 kDa) (manufactured by Thermo Fisher). The third solution was used as the dialysate. After dialysis, the solution inside the dialysis membrane (containing proteinbound nanoparticles) 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]

[0095] In Manufacturing Condition 1-1 above, the process involved mixing the first and second solutions — dilution — addition of the protein solution — dialysis — concentration —> separation. The addition of the protein solution began 30 minutes after obtaining the first mixture of the first and second solutions. Dialysis was initiated 30 minutes after adding the protein solution.

[0096] In contrast, Manufacturing Condition 1-2 followed a different sequence: mixing the first and second solutions —> dilution — > dialysis — concentration — addition of the protein solution —> separation. The addition of the protein solution in this condition was performed 18 hours after obtaining the first mixture.

[0097] In Manufacturing Condition 1-2, the first solution was mixed at a flow rate of 2.25 mL / min and the second solution was mixed at a flow rate of 6.75 mL / min at 25°C to prepare the first mixture, using the Automated Nanoparticle System (manufactured by Particle Works). The obtained first mixture was diluted with an equal volume of the third solution to obtain a diluted first mixture. Thirty minutes after obtaining the first mixture, dialysis of the diluted first mixture was initiated. The dialysis was conducted at 4°C for 16 hours using a Slide-a-Lyzer G3 dialysis membrane (10 kDa) (manufactured by Thermo Fisher). After dialysis, the solution inside the dialysis membrane (containing protein-boundNDJP.025WO PATENTnanoparticles) was concentrated to approximately 1 mL using Amicon Ultra-15 100 kDa (manufactured by Merck Millipore). Eighteen hours after obtaining the first mixture, the protein solution was added to the concentrated solution containing protein-bound nanoparticles and mixed. The molar ratio of added protein to maleimide was adjusted to 0.1. The solution was gently stirred at 4°C for 16 hours on a shaker (manufactured by VWR Signature 3D Rotator Waver; VWR), and the resulting solution was subjected to the separation process.(Separation Process)[Manufacturing Condition 2-1]

[0098] To separate protein-bound lipid nanoparticles, Q Sepharose FF (Fast Flow) (manufactured by Cytiva) was used as the resin. A column packed with the resin in Econo-pac Chromatography Columns (manufactured by Bio-Rad) was used for the separation. The third solution was used as the running buffer. After equilibrating the column packed with 15 mL of Q Sepharose FF by flushing it with the third solution for over an hour, a sample containing protein-bound lipid nanoparticles was added to the top of the column. The solution eluted 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.[Manufacturing Condition 2-2]

[0099] Manufacturing Condition 2-2 was conducted in the same manner as Manufacturing Condition 2-1 , except that Sepharose CL6B was used instead of Q Sepharose FF (manufactured by Cytiva) as the resin.NDJP.025WO PATENTAnalytical Conditions)[N / P Ratio]

[0100] The N / P ratio (nitrogen / phosphorus ratio) is the molar ratio of nitrogen atoms (N) in the lipid to phosphate groups (P) in the nucleic acid molecule. This ratio serves as an indicator to evaluate the electrostatic interactions between nucleic acids and cationic components. Here, the nitrogen atoms (N) refer to the total number of nitrogen atoms, such as those in amine groups, present in cationic lipids or ionizable cationic lipids. The phosphate groups (P) refer to the total number of phosphate groups present 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 cationic lipids or ionizable cationic lipids by the molar amount of phosphate groups in the nucleic acid.[Measurement ofZ-Average Particle Size and PDI]

[0101] 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 * PBS. The measurements were conducted with a Zetasizer Nano ZS (manufactured by Malvern).[Measurement ofmRNA Encapsulation Efficiency]

[0102] The mRNA encapsulation efficiency in protein-bound lipid nanoparticles was measured using the Ribogreen reagent (manufactured by Invitrogen).

[0103] The concentration of nucleic acids outside the protein-bound lipid nanoparticles was determined using a test solution (Solution X), which was prepared byNDJP.025WO PATENTdiluting 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.

[0104] 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 wavelength of 485 nm and an emission wavelength of 528 nm. The nucleic acid concentration was calculated using a calibration curve.

[0105] The mRNA encapsulation efficiency (%) was calculated using the following formula:n»RNA Eneaps*>kitoa E&iciwy / Nur It ic Avul CoJicejiuatliHi in MciJSTticifiem Solntic-ii Y Nucleic Acid in Meais nf’riiieui Soliitic'ij X \■ i0(’\ Nucleic Acid Cfjnvwitfatitm in Svhiiiofj Y / [Measurement of mRNA Recovery Rate]

[0106] 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.

[0107] The mRNA recovery rate (%) was calculated using the following formula:NDJP.025WO PATENTR A Hew very Rate %} ~of Niidek Acids iti th" Proteiii-Bounr! Lipid Nauorwsrtieie Solution afti’T the Separation Process h\ Amonnt oi Ntideie Aesds in the Second Solution Issea tor Portitie Formation /

[0108] A high mRNA recovery rate indicates that a significant portion of the input mRNA was efficiently incorporated into the protein-bound lipid nanoparticles during the particle manufacturing process and successfully recovered after the separation process.

[0109] In the particle manufacturing process, a high mRNA recovery rate indicates efficient nucleic acid encapsulation into the lipid nanoparticles and effective protein conjugation. In the separation process, it demonstrates efficient separation and purification of the protein-bound lipid nanoparticles containing encapsulated mRNA.

[0110] Conversely, a low mRNA recovery rate indicates potential challenges in either the particle manufacturing process, the separation process, or both:

[0111] In the particle manufacturing process, it may indicate reduced efficiency in generating protein-bound lipid nanoparticles.

[0112] In the separation process, it may point to inefficiencies in separating and purifying the nanoparticles, resulting in insufficient recovery of mRNA-containing particles.

[0113] Thus, a high mRNA recovery rate serves as an indicator of good performance in both the particle manufacturing and separation processes during the production of protein-bound lipid nanoparticles.30% or higher: Indicates a good process.50% or higher: Indicates a highly efficient process.NDJP.025WO PATENT[Analysis of Protein-Lipid Conjugates]

[0114] 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.

[0115] 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.

[0116] This analysis confirmed whether proteins are chemically bound to lipid nanoparticles. If proteins form protein-lipid conjugates, the protein bands shift toward the higher molecular weight side in the gel electrophoresis results. In contrast, no shift is observed if the protein-lipid conjugates are not formed.

[0117] Table 2 below presents the detection results for conjugated antibodies. Results marked as “Yes” indicate the formation of protein-lipid conjugates, demonstrating that chemically bonded protein-bound lipid nanoparticles were successfully obtained.[Apparent Surface pKa of Protein-Bound Lipid Nanoparticles]

[0118] The apparent pKa of the surface of protein-bound lipid nanoparticles was measured under the conditions described by Jayaramanno et al. "Maximizing theNDJP.025WO PATENTPotency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo" (Angewandte Chemie Volume 124, Issue 34, 8657-8661 , 2012).

[0119] The protein-bound lipid nanoparticles were diluted in a buffer solution containing 10 mM HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid), 10 mM MES (2-(N-morpholino)ethanesulfonic acid), 10 mM ammonium acetate, and 130 mM NaCIto achieve a lipid concentration of 100 pM. ApH range from 3.0 to 12.0 was prepared, and TNS (p-toluidino-6-naphthalene sulfonic acid) was added to achieve a final concentration of 1 pM. After mixing, the fluorescence intensity was measured using a Synergy 2 spectrophotometer (manufactured by Biotek) at 25°C with an excitation wavelength of 321 nm and an emission wavelength of 445 nm.

[0120] A sigmoidal best-fit analysis was applied to the fluorescence data, and the apparent surface pKa was determined as the pH value corresponding to half-maximal fluorescence intensity.[Evaluation]

[0121] When the mRNA recovery rate exceeds 30% and the detection result for the conjugated antibody is "Yes," it indicates that the protein-bound lipid nanoparticles with chemically bound proteins were efficiently produced. On the other hand, if the mRNA recovery rate exceeds 30% but the conjugated antibody detection result is "No," it suggests that the proteins were not chemically bound to the lipid nanoparticle surface. In such cases, the chemical conjugation via maleimide during the particle manufacturing process was insufficient, resulting in suboptimal nanoparticles.NDJP.025WO PATENT

[0122] Furthermore, if the mRNA recovery rate is below 30%, the production of protein-bound lipid nanoparticles was inadequate, regardless of the conjugated antibody detection results.[Results]

[0123] As shown in Table 2 below, in Examples 1 to 3, which employed the combination of Manufacturing Condition 1-1 and Manufacturing Condition 2-1 , the mRNA recovery rate exceeded 30%, and conjugated antibodies were detected.

[0124] In Example 4, Manufacturing Condition 1-1 was combined with the conventional Manufacturing Condition 2-2. While Manufacturing Condition 1-1 enabled the generation of protein-bound lipid nanoparticles, the separation and recovery efficiency for nanoparticles with high apparent pKa values was low under Manufacturing Condition 2-2, leading to a reduced mRNA recovery rate. Although Example 4 successfully produced protein-bound lipid nanoparticles with high apparent pKa, Example 3, which employed Manufacturing Condition 2-1, showed superior results in terms of separation and recovery efficiency.

[0125] In Comparative Example 1 , which did not adopt Manufacturing Condition 2-1 , the mRNA recovery rate was significantly low. Due to the use of Manufacturing Condition 2-2 in the separation process, protein-bound lipid nanoparticles could not be effectively separated and recovered (particle yield was below 1%), making the detection of conjugated antibodies impossible.

[0126] In Comparative Example 2, which did not adopt Manufacturing Condition 1-1 , the mRNA recovery rate was high; however, conjugated antibodies wereNDJP.025WO PATENTnot detected, indicating that nanoparticles with chemically bound proteins were not obtained.

[0127] For Compositions 1 and 2, which included cationic lipids with quaternary ammonium structures, it was found that the cysteine-binding capability of maleimide rapidly diminished. For such formulations, conventional methods like Manufacturing Condition 1-2 rendered chemical conjugation difficult. However, using a method such as Manufacturing Condition 1-1, where the protein solution is promptly reacted with maleimide, enabled successful chemical conjugation. Additionally, for Compositions 1 and 2, adopting Manufacturing Condition 2-1 with Q Sepharose as the resin allowed effective separation and purification.

[0128] From these results, it was confirmed that combining Manufacturing Condition 1-1 and Manufacturing Condition 2-1 enables the efficient production of proteinbound lipid nanoparticles with high apparent surface pKa values.NDJP.025WO PATENTTABLE 2Manufacturing Z-Average PDI mRNA mRNA Conjugated Apparent Condition and Particle Encapsulation Recovery Antibody Surface Lipid Size (nm) Efficiency (%) Rate (%) Detection pKa Composition1-1Example 1 2-1 90 0.155 >99 53 Yes 9.49Composition 11-1Example 2 2-1 69 0.179 >99 45 Yes 9.25Composition 21-1Example 3 2-1 61 0228 98 39 Yes 757Composition 31-1Comparative2-2 102 0.032 51 <1 - — Example 1Composition 21-2Comparative2-1 85 0.134 >99 50 No 9.20 Example 2Composition 21-1Example 4 2-2 103 0.101 >99 <20 Yes 7.31Composition 3

Claims

NDJP.025WO PATENTWHATIS CLAIMED IS:

1. A protein-bound lipid nanoparticle, wherein an apparent pKa of the surface of the protein-bound lipid nanoparticle is 7.0 to 10.0, and the apparent pKa of the surface is measured by a TNS (p-toluidino-6-naphthalene sulfonic acid) measurement method.

2. The protein-bound lipid nanoparticle according to claim 1, comprising a cationic lipid or an ionizable cationic lipid, a phospholipid, a sterol, a polyethylene glycol(PEG)-modified lipid, and a conjugate of protein and lipid, and encapsulating a nucleic acid.

3. A method of manufacturing the protein-bound lipid nanoparticle according to claim 1 or 2, comprising:(a) mixing a first solution and a second solution to obtain a first mixed solution;(b) mixing the first mixed solution and an unbound protein either simultaneously with said (a) or within an hour after completion of said (a) to obtain a second mixed solution; and(c) dialyzing, filtering, and / or diluting the second mixed solution either simultaneously with said (b) or within an hour after completion of said (b), wherein the first solution comprises ethanol and a lipid having a maleimide group in its molecule, said (b) being conducted under conditions selected to chemically bind the unbound protein to the lipid,the second solution comprises a nucleic acid and a buffer, and the unbound protein comprises a thiol group.NDJP.025WO PATENT4. A method of manufacturing the protein-bound lipid nanoparticle according to claim 1 or 2, comprising:separating the protein-bound lipid nanoparticle and the unbound protein from a solution containing the protein-bound lipid nanoparticle and the unbound protein,wherein said separating comprises a gel filtration using a running buffer and a column packed with a resin, andthe resin is selected from the group consisting of a strong basic anion exchange resin and a weak basic anion exchange resin.

5. A method of manufacturing a protein-bound lipid nanoparticle, comprising:(a) mixing a first solution and a second solution to obtain a first mixed solution;(b) mixing the first mixed solution and an unbound protein either simultaneously with said (a) or within an hour after completion of said (a) to obtain a second mixed solution; and(c) dialyzing, filtering, and / or diluting the second mixed solution either simultaneously with said (b) or within an hour after completion of said (b), wherein the first solution comprises a lipid having a maleimide group in its molecule, said (b) being conducted under conditions selected to chemically bind the unbound protein to the lipid,the second solution comprises a nucleic acid,the unbound protein comprises a thiol group, andan apparent pKa of the surface of the protein-bound lipid nanoparticle is 7.0 to 10.0.NDJP.025WO PATENT6. The method according to claim 5, further comprising:separating the protein-bound lipid nanoparticle and the unbound protein from a solution containing the protein-bound lipid nanoparticle and the unbound protein,wherein said separating comprises a gel filtration using a running buffer and a column packed with a resin,wherein the resin is selected from the group consisting of a strong basic anion exchange resin and a weak basic anion exchange resin.

7. The method according to claim 6, wherein the resin is DEAE (diethylaminoethyl cellulose) or Q-Sepharose.

8. The method according to any one of claims 5 to 7, wherein the first solution further comprises ethanol.

9. The method according to any one of claims 5 to 8, wherein the second solution further comprises a buffer.

10. The method according to any one of claims 5 to 9, wherein the apparent pKa of the surface of the protein-bound lipid nanoparticle is measured by a TNS (p-toluidino-6-naphthalene sulfonic acid) measurement method.

11. The method according to any one of claims 5 to 10, wherein the unbound protein is an antibody or an antigen-binding fragment thereof.

12. The method according to claim 11, wherein the antibody is an anti-CD8 antibody or an anti-human transferrin receptor antibody, wherein the anti-CD8 antibody comprises an amino acid sequence represented by Sequence ID No. 1 as a heavy chainNDJP.025WO PATENTand an amino acid sequence represented by Sequence ID No. 2 as a light chain, and the anti-human transferrin receptor antibody comprises an amino acid sequence represented by Sequence ID No. 3 as a heavy chain and an amino acid sequence represented by Sequence ID No. 4 as a light chain.

13. The method according to any one of claims 5 to 12, wherein the first mixed solution is mixed with the unbound protein within 30 minutes after the completion of said (a) to obtain the second mixed solution.

14. The method according to one of claims 5 to 13, wherein the apparent pKa of the surface of the protein-bound lipid nanoparticle is 9.0 to 10.0.

15. The method according to any one of claims 5 to 14, wherein the proteinbound lipid nanoparticle comprises a second lipid selected from the group consisting of a cationic lipid, an ionizable cationic lipid, a phospholipid, a sterol, and a polyethylene glycol(PEG)-modified lipid.

16. The method according to claim 15, wherein the cationic lipid is selected from the group consisting of DOTAP (1 ,2-dioleoyl-3-trimethylammonium-propane), DC-Chol (3p-[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), CTAB (cetyltrimethylammonium bromide), and HEDC (2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethan-aminium).NDJP.025WO PATENTThe method according to claim 15, wherein the ionizable cationic lipid is selected from the group consisting of DLin-MC3-DMA (1 ,2-dilinoleoyl-sn-glycero-3-methyl dimethylamine), DODAP (1,2-dioleoyl-3-dimethylaminopropane), DDA (dimethyldioctadecylammonium), C12-200 (1 ,1'-[[(2-(dimethylamino)ethyl)imino]bis-butanoyl]ethyl methylamine), OF-Deg-Lin (oxyfluoroalkyldeglinolein), ATLAS-75 (acylthiolipid amine 75), DLin-KC2-DMA (1 ,2-dilinoleoyl-sn-glycero-3-dimethylaminoethanol), DLin-DMA (1,2-dilinoleoyl-sn-glycero-3-dimethylamine), ALC-0315 ([(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyl-decanoate)), SM-102 ([(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(9-heptadecenoate)),and Compound 3:Compound 3.

18. The method according to claim 15, wherein the phospholipid is selected from the group consisting of 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-phosphoglycerol), DSPG (1 ,2-distearoyl-sn-glycero-3-phosphoglycerol), DPPG (1 ,2-dipalmitoyl-sn-glycero-3-phosphoglycerol), 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-phosphoric acid),NDJP.025WO PATENTDPPA (1 ,2-dipalmitoyl-sn-glycero-3-phosphoric acid), and DSPA (1 ,2-distearoyl-sn-glycero-3-phosphoric acid).

19. The method according to claim 15, wherein the PEG-modified lipid is selected from the group consisting of mPEG-DSPE (methoxy-PEG-distearoylphosphatidylethanolamine), mPEG-DMG (methoxy-PEG-dimyristoylglycerol), mPEG-DOPE (methoxy-PEG-dioleoylphosphatidylethanolamine), mPEG-Ceramide (C14, C16, C18), mPEG-Cholesterol, and mPEG-DSG (methoxy-PEG-distearoylglycerol).

20. The method according to any one of claims 5 to 14, wherein the second mixed solution comprises a lipid mixture selected from the group consisting of: (i) Compound 1 , cholesterol, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-[carbonyl-methoxypolyethylene glycol]-1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), Compound 2, and DSPE-PEG-maleimide, and (ii) Compound 3, cholesterol, DOPE, N-[carbonyl-methoxypolyethylene glycol]- 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), Compound 2, and DSPE-PEG-maleimide,wherein said compounds 1-3 are represented a chemical formula below:Compound 1 :Compound 2 :NDJP.025WO PATENTCompound s :

21. A method of manufacturing a protein-bound lipid nanoparticle, comprising:separating a protein-bound lipid nanoparticle and an unbound protein from a solution containing the protein-bound lipid nanoparticle and the unbound protein, wherein said separating comprises a gel filtration using a running buffer and a column packed with a resin,wherein the resin is selected from the group consisting of a strong basic anion exchange resin and a weak basic anion exchange resin when an apparent pKa of the surface of the protein-bound lipid nanoparticle is the same as, within a range of ±0.2 of, or higher than a pH of the running buffer, andthe resin is selected from the group consisting of a strong acidic cation exchange resin and a weak acidic cation exchange resin when the apparent pKaNDJP.025WO PATENTof the surface of the protein-bound lipid nanoparticle is lower than the pH of the running buffer.

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