Lipid nanoparticle manufacturing method and manufacturing device thereof

Abstract

The present invention relates to a method and an apparatus for producing lipid nanoparticles, and does not require a separate process for selecting the produced lipid nanoparticles, thereby increasing the production yield in the subsequent sterilization and filtration process, etc. In addition, it is possible to produce lipid nanoparticles of uniform size by reducing the content of ionized lipids that are toxic when injected into the body, which is different from the conventional optimal drug and lipid ratio.

Description

[Technical field]

[0001] The present invention relates to a method and an apparatus for producing lipid nanoparticles (LNPs). [Background technology]

[0002] Lipid nanoparticles (LNPs) are effective drug delivery systems for biologically active compounds such as therapeutic nucleic acids, proteins, and peptides that are cell impermeable.

[0003] Vaccines are usually subdivided into "first generation", "second generation" and "third generation" vaccines, with genetic vaccines, i.e. vaccines for genetic vaccination, usually being understood as "third generation" vaccines. Genetic vaccines typically consist of genetically engineered nucleic acid molecules that allow the expression in vivo of peptides or protein (antigen) fragments characteristic of pathogens or tumor antigens. Genetic vaccines are expressed upon administration to a patient and after uptake by target cells. Expression of the administered nucleic acid leads to the production of the encoded protein. If such a protein is recognized as foreign by the patient's immune system, an immune response is triggered.

[0004] From the perspective of genetic vaccination, not only DNA but also RNA can be used as the nucleic acid molecule for administration, DNA being known to be relatively stable and easy to handle.

[0005] However, the use of DNA carries the risk of unwanted insertion of the administered DNA fragment into the patient's genome, potentially resulting in mutagenic events such as loss of function of the damaged gene.

[0006] By using RNA instead of DNA for gene vaccination, the risk of unwanted genome integration and the generation of anti-DNA antibodies is minimized or prevented, but RNA can be easily degraded by ubiquitous RNases and is quite unstable, and has problems of impermeability, fragility and immunogenicity.

[0007] Although many developments have been made in the past few years, lipid nanoparticle formulations have been used as an efficient method for administration of mRNA vaccines capable of eliciting adaptive immune responses.

[0008] The lipid nanoparticle formulations can improve the delivery of nucleic acids in vivo.

[0009] The lipid nanoparticle-based drug delivery system is a multi-component formulation containing ionizable lipids, non-ionizable lipids, neutral lipids and fusogenic lipids. The anionic ionizable lipids bind to the cationic nucleic acid, while the other components support the stable self-assembly of lipid nanoparticles.

[0010] The lipid nanoparticles can be manufactured with an optimal drug:lipid ratio to protect the nucleic acid from degradation and clearance by serum, be suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid.

[0011] When the lipid nanoparticles are prepared by the conventional method, the particles are not uniform in size, and a separate classification process is required, resulting in reduced production efficiency.

[0012] In order to solve these problems and improve production efficiency, it is necessary to develop a manufacturing process for lipid nanoparticles with a uniform diameter. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Korean Patent Publication No. 10-2019-0093816 Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide a method and an apparatus for producing lipid nanoparticles.

[0015] Another object of the present invention is to provide an apparatus for producing lipid nanoparticles, which does not require a separate process for selecting the produced lipid nanoparticles, thereby increasing the production yield in the subsequent sterilization filtration process, etc.

[0016] Another object of the present invention is to provide a method for producing lipid nanoparticles of uniform size that deviates from the conventional optimal drug and lipid ratio, reduces the content of ionized lipids that cause toxicity problems when injected into the body, and [Means for solving the problem]

[0017] In order to achieve the above object, the present invention relates to a method for producing lipid nanoparticles, which may include the steps of: producing an aqueous phase solution containing nucleic acid; dissolving an ionizable lipid in an organic solution to produce a first oil phase solution; dissolving a non-ionizable lipid, a neutral lipid, and a fusogenic lipid in an organic solution to produce a second oil phase solution; injecting the aqueous phase solution into a first channel and allowing it to flow; injecting the first oil phase solution into a second channel and allowing it to flow; the aqueous phase solution and the first oil phase solution cross each other and flow through a stirring channel as a first mixed solution; flowing the second oil phase solution into a third channel connected to the stirring channel, crossing the first mixed solution and mixing it with the first mixed solution as a second mixed solution; and passing the second mixed solution through a stirring section in the stirring channel to form lipid nanoparticles containing nucleic acid.

[0018] The stirring channel may include stirred and non-stirred mixing modules.

[0019] The mixing module may be formed in a plurality of mixing modules in the stirring channel, and the n-th mixing module may be formed in order based on the fluid flow direction of the stirring channel, and the n-th mixing module may refer to the order of the mixing modules repeatedly formed in the stirring channel.

[0020] The third channel may form an intersection between the first mixing module to the fifth mixing module and be connected to the stirring channel, so that the first mixed solution and the second oil phase solution intersect to form a laminar flow.

[0021] The lipid nanoparticles may be uniformly spherical with a polydispersity index of 0.2.

[0022] The ionizable lipid may be contained in an amount of 10 to 30 mol % based on the total weight of lipids in the lipid nanoparticle.

[0023] The weight ratio of the ionizable lipid and nucleic acid may be from 3:1 to 50:1.

[0024] The nucleic acid may be selected from the group consisting of RNA, DNA, siRNA (short interfering RNA), mRNA (messenger RNA), aptamer, antisense ODN (antisense oligodeoxynucleotide), antisense RNA, ribozyme, DNAzyme, and mixtures thereof.

[0025] The non-ionizable lipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleolphosphatidyl ethanolamine (DOPE), bis(diphenylphosphino)ethane (DPPE), diacyl phosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylserine, and mixtures thereof.

[0026] The neutral lipid may be selected from the group consisting of polyethylene glycol 2000 distearoylphosphatidylethanolamine (PEG(2000)DSPE), DMG-PEG, PEG-DMPE, DPPE-PEG, DPG-PEG, PEG-DOPE, and mixtures thereof.

[0027] The fusogenic lipid may be selected from the group consisting of phospholipids, cholesterol, tocopherol and mixtures thereof.

[0028] Lipid nanoparticles containing a low concentration of ionized lipid according to another embodiment of the present invention may be prepared by the above-mentioned preparation method.

[0029] Another embodiment of the present invention relates to an apparatus for manufacturing lipid nanoparticles containing a low concentration of ionized lipids, the apparatus comprising: a first channel for flowing an aqueous phase solution containing nucleic acid; a second channel for flowing a first oil phase solution containing ionizable lipids; a third channel for flowing a second oil phase solution containing non-ionizable lipids, neutral lipids and fusogenic lipids; and a stirring channel, wherein the first channel and the second channel form a first intersection, the intersection being connected to the stirring channel, and the stirring channel may comprise a mixing module of a stirring section and a non-stirring section.

[0030] The mixing module may be formed in a plurality of mixing modules in the stirring channel, and the n-th mixing module may be formed in order based on the fluid flow direction of the stirring channel, and the n-th mixing module may refer to the order of the mixing modules repeatedly formed in the stirring channel.

[0031] There may be between 3 and 70 mixing modules within the mixing channel.

[0032] The third channel may be coupled to the stirring channel, forming an intersection between the first mixing module to the fifth mixing module.

[0033] The length of the mixing module may be 1 to 5 mm based on the flow direction of the fluid in the stirring channel.

[0034] The length ratio of the stirring portion and the non-stirring portion may be 45:1 to 5:0.3 based on the flow direction of the fluid in the stirring channel.

[0035] The stirring portion may have a groove formed therein to mix the inflowing fluid.

[0036] The grooves of the stirring portion may induce chaotic mixing to enhance the mixing efficiency of laminar flow in the stirring channel.

[0037] The shape of the groove may be square, semicircular or triangular. Effect of the Invention

[0038] Since the present invention does not require a separate process for selecting the produced lipid nanoparticles, the production yield can be increased in the subsequent sterilization filtration process and the like.

[0039] In addition, it is possible to deviate from the conventional optimal drug / lipid ratio, reduce the content of ionized lipids that pose a toxicity problem when injected into the body, and produce lipid nanoparticles of uniform size. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 is a diagram of a manufacturing apparatus according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a diagram of a conventional apparatus for producing lipid nanoparticles according to one embodiment of the present invention. [Diagram 3] FIG. 3 is a photograph of a manufacturing apparatus according to an embodiment of the present invention. [Figure 4] FIG. 4 shows the PDI measurement results of lipid nanoparticles prepared according to one embodiment of the present invention. [Diagram 5]FIG. 5 is a Cryo-EM photograph of lipid nanoparticles according to one embodiment of the present invention. [Figure 6] FIG. 6 shows the results of measuring the mixing index rate (%) according to the flow rate ratio of the aqueous phase solution and the first oil phase solution in one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention relates to a method for producing lipid nanoparticles, the method including the steps of: producing an aqueous phase solution containing nucleic acid; dissolving an ionizable lipid in an organic solution to produce a first oil phase solution; dissolving a non-ionizable lipid, a neutral lipid, and a fusogenic lipid in an organic solution to produce a second oil phase solution; injecting and flowing the aqueous phase solution into a first channel; injecting and flowing the first oil phase solution into a second channel; the aqueous phase solution and the first oil phase solution cross each other to flow through a stirring channel as a first mixed solution; flowing the second oil phase solution into a third channel connected to the stirring channel, crossing the first mixed solution and mixing it with the first mixed solution to form a second mixed solution; and passing the second mixed solution through a stirring section in the stirring channel to form lipid nanoparticles containing nucleic acid.

[0042] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to exemplary embodiments thereof, so that those skilled in the art can easily practice the present invention. However, the present invention may be embodied in many different forms and should not be construed as being limited to the exemplary embodiments set forth herein.

[0043] mRNA is an abbreviation for messenger ribonucleic acid. DNA containing genetic information becomes mRNA, which is used to synthesize proteins, and is an intermediate that connects DNA and proteins.

[0044] The COVID-19 pandemic has sparked interest and development in mRNA vaccines. mRNA vaccines have several advantages over other types of vaccines. The biggest advantage of mRNA vaccines is that lipid nanoparticles (LNPs) containing mRNA are a platform technology, allowing for rapid technological development to combat viruses that mutate frequently, such as COVID-19.

[0045] Specifically, a protective protein antigen can be identified, and the gene for the antigen can be sequenced to produce mRNA. When new mRNA is produced using this method and the formulation design and manufacturing process of conventional mRNA vaccines are used, it is possible to rapidly produce an mRNA vaccine. This means that because mRNAs encoding different antigens are very similar chemically and physically, the formulation design and manufacturing process of the new mRNA vaccine can be carried out using the same steps as the formulation and manufacturing process of conventional mRNA vaccines.

[0046] Due to the negative charge of the phosphate groups, mRNA is a polycationic macromolecule in the pH range generally used for parenteral administration. As described above, the electrical properties of the negatively charged mRNA can be used to prepare lipid nanoparticles using positively charged ionized lipids (ionisable lipids or cationic lipids). Specifically, the ionized lipids are positively charged lipids that bind strongly to the negatively charged mRNA through electrical attraction. In addition to the ionized lipids, non-ionizable lipids, neutral lipids and fusogenic lipids are further included to form lipid nanoparticles.

[0047] The nucleic acid-lipid particles of U.S. Patent No. 9,364,435 are disclosed as comprising (a) a nucleic acid, (b) anionic lipids, (c) non-anionic lipids, and (d) fusogenic lipids, and the anionic lipids are contained in an amount of 50 mol% to 85 mol%, the non-anionic lipids in an amount of 13 mol% to 49.5 mol%, and the fusogenic lipids in an amount of 0.5 mol% to 2 mol%, based on the total lipid content in the particle.

[0048] Furthermore, the nucleic acid-lipid particles in European Patent No. 2279254 are disclosed to contain, based on the total lipid content within the particles, 50 mol % to 65 mol % of the anionic lipid, 49.5 mol % or less of the non-anionic lipid, 30 mol % to 40 mol % of cholesterol or a derivative thereof, and 0.5 mol % to 2 mol % of a fusogenic lipid.

[0049] As described above, nanolipid particles containing mRNA have been confirmed to contain a large amount of ionized lipids (anionic lipids) within the particles.

[0050] However, as mentioned above, there are still some concerns about the safety of mRNA-containing lipid nanoparticles (LNPs) for therapeutic use due to in vitro and in vivo toxicity. Such toxicity is mainly caused by non-specific charge interactions. In other words, the toxicity of positively charged ionized lipids has become an issue, and research to address this issue is ongoing.

[0051] The most intuitive approach would be to try to reduce the amount of anionic ionized lipid used, but when using low concentrations of ionized lipid below the limit, there is a problem that the size of the lipid nanoparticles (LNPs) produced is not uniform. When producing lipid nanoparticles (LNPs) with non-uniform sizes, an additional process is required to remove lipid nanoparticles (LNPs) of undesired sizes, which leads to large losses in the subsequent sterilization filtration process, etc. This causes a significant decrease in the production yield of lipid nanoparticles containing mRNA.

[0052] The present invention relates to a method for producing lipid nanoparticles containing nucleic acids, and aims to provide a production method capable of producing lipid nanoparticles with a uniform particle size.

[0053] As described above, the present invention is characterized in that it produces lipid nanoparticles having a uniform diameter while reducing the content of ionized lipids, which are problematic in terms of toxicity. Even if the content of ionized lipids is within the range of the lipid content for producing conventional lipid nanoparticles or the range of the ionized lipid content is reduced, it is characterized in that it is possible to produce particles having a uniform diameter by using a method different from the conventional production method.

[0054] Specifically, the method may include the steps of: preparing an aqueous phase solution containing nucleic acid; dissolving an ionizable lipid in an organic solution to prepare a first oil phase solution; dissolving a non-ionizable lipid, a neutral lipid, and a fusogenic lipid in an organic solution to prepare a second oil phase solution; injecting and flowing the aqueous phase solution into a first channel; injecting and flowing the first oil phase solution into a second channel; the aqueous phase solution and the first oil phase solution cross each other and flow through a stirring channel as a first mixed solution; flowing the second oil phase solution into a third channel connected to the stirring channel, where it crosses with the first mixed solution and is mixed with the second mixed solution; and the second mixed solution passing through a stirring section in the stirring channel to form lipid nanoparticles containing nucleic acid.

[0055] Specifically, the nucleic acid is mixed with a solvent to prepare an aqueous solution. The solvent is not limited to the above-mentioned examples, such as a citric acid solution having a pH of 3.0, and any solvent capable of producing lipid nanoparticles by mixing with the nucleic acid can be used without any restrictions.

[0056] The nucleic acid may be selected from the group consisting of RNA, DNA, siRNA (short interfering RNA), mRNA (messenger RNA), aptamer, antisense ODN (antisense oligodeoxynucleotide), antisense RNA, ribozyme, DNAzyme, and mixtures thereof, and is preferably mRNA and is not limited to the above examples.

[0057] The nucleic acid is used for preventing or treating a disease, and for example, it is used to synthesize a spike protein to combat the COVID-19 virus, such as a COVID-19 vaccine. The nucleic acid is not limited to the above examples, and any nucleic acid for preventing or treating a disease can be used.

[0058] Then, the ionized lipid is dissolved in the organic solution to prepare a first oil phase solution. The ionized lipid may be ALC-0315 (Genevant), ALC-0159 (Genevant), DLinDAP, Dlin-MC3-DMA, or SM102 (Arbutus). The ionized lipid is not limited to the examples, and any ionized lipid used in the preparation of lipid nanoparticles may be used without limitation.

[0059] The organic solution is an alcohol, specifically, methanol, ethanol, isopropanol, n-propanol, etc., and is preferably ethanol, but is not limited to the above examples, and any organic solvent capable of uniformly dissolving ionized lipids can be used without limitation.

[0060] A second oil phase solution is prepared by dissolving a non-ionizable lipid, a neutral lipid and a fusogenic lipid in an organic solution.

[0061] The non-ionized lipid may be included together with the fusogenic lipid to enhance the safety of the lipid nanoparticle. Although the lipid nanoparticle is intended to deliver nucleic acid to a target tissue or organ, it has a problem that it is destroyed before reaching the target tissue or organ after being injected into the body. In order to prevent such a problem, the non-ionized lipid and the fusogenic lipid may be included. Specifically, the non-ionized lipid is selected from the group consisting of DSPC (distearoylphosphatidylcholine), DOPE (dioleolphosphatidyl ethanolamine), DPPE (bis(diphenylphosphino)ethane), diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylserine, and mixtures thereof, and is preferably DSPC, but is not limited to the above examples.

[0062] The fusogenic lipid may be selected from the group consisting of cholesterol, tocopherol and mixtures thereof, preferably cholesterol and not limited to the above examples.

[0063] The neutral lipid is included to adjust the particle size and to act as a steric barrier to prevent aggregation during storage, and may be specifically selected from the group consisting of polyethylene glycol 2000 distearoylphosphatidylethanolamine (PEG(2000)DSPE), DMG-PEG, PEG-DMPE, DPPE-PEG, DPG-PEG, PEG-DOPE, and mixtures thereof, preferably DMG-PEG, but is not limited to the above examples.

[0064] The organic solution used to prepare the second oil phase solution is an alcohol, and specifically may be methanol, ethanol, isopropanol, n-propanol, etc., but is preferably ethanol, but is not limited to the above examples, and any organic solvent capable of uniformly dissolving ionized lipids can be used without limitation.

[0065] The prepared aqueous phase solution and first oil phase solution are injected into a first channel and a second channel, which form an intersection and are connected to a mixing channel, as described below.

[0066] The aqueous phase solution and the first oil phase solution injected into the first and second channels, respectively, form a laminar flow at the intersection and flow through the stirring channel.

[0067] In general, the process of preparing lipid nanoparticles involves injecting an aqueous solution containing dissolved nucleic acid and an oily solution containing dissolved four types of lipids into each channel, and the aqueous solution and the oily solution injected into the channel form a laminar flow at the intersection and flow through the stirring channel. The aqueous solution and the oily solution injected into the stirring channel are mixed, and the nucleic acid in the aqueous solution and the ionized lipids, non-ionized lipids, neutral lipids and fusogenic lipids in the oily solution are bound by electrostatic attraction to form lipid nanoparticles.

[0068] That is, lipid nanoparticles are prepared by mixing an aqueous phase solution and an oil phase solution, and the oil phase solution contains a mixture of all four types of lipids.

[0069] On the other hand, the method for producing lipid nanoparticles containing a low concentration of ionized lipids of the present invention is characterized in that the oil phase solution is divided into a first oil phase solution and a second oil phase solution, the first oil phase solution containing only ionized lipids, and the second oil phase solution containing the remaining lipids.

[0070] As described above, the aqueous phase solution flows through the first channel and the first oil phase solution flows through the second channel, and the nucleic acids in the aqueous phase solution and the ionized lipids in the first oil phase solution preferentially bind due to electrostatic attraction.

[0071] In order to allow the nucleic acids in the aqueous phase solution and the ionized lipids in the first oil phase solution to bind more easily by electrostatic attraction, they are passed through a stirring section in a stirring channel.

[0072] The stirring channel specifically includes a plurality of mixing modules each including a stirring section and a non-stirring section.

[0073] The mixing modules are formed in an n-th order based on the fluid flow direction of the mixing channel, and the n-th mixing module refers to the order of the mixing modules repeatedly formed in the mixing channel.

[0074] Specifically, the first mixing module may be formed at the forefront based on the fluid flow direction of the stirring channel, and the second mixing module, the third mixing module, etc. may be formed thereafter.

[0075] The mixing module includes a stirring section and a non-stirring section. As described below, the stirring section is characterized by having a groove formed therein to allow the aqueous phase solution and the first oil phase solution to be mixed well. The aqueous phase solution and the first oil phase solution flow in a laminar flow, and become a chaotic mix in the stirring section, and the nucleic acid and the ionized lipid are combined through the mixing process.

[0076] The nucleic acid is specifically mRNA, which is cationic as explained above, and the ionized lipid is anionic, and they are bound together by electrostatic attraction between them.

[0077] The aqueous phase solution and the first oil phase solution pass through the stirring channel to carry out the mixing process, and as they flow through the stirring channel as the first mixed solution, the second mixed solution is sequentially injected into the third channel and mixed with the first mixed solution in the stirring channel.

[0078] The third channel is connected to the mixing channel by forming an intersection between the first mixing module and the fifth mixing module. Specifically, the third channel is connected to the mixing channel by forming an intersection between the second mixing module and the fourth mixing module, and more preferably, the third channel is connected to the mixing channel by forming an intersection in a non-mixing portion of the third mixing module.

[0079] As described above, the binding portion of the third channel was determined taking into consideration the degree of binding between the nucleic acid and the ionized lipids in order to bind the non-ionized lipids, neutral lipids and fusogenic lipids of the second oil phase channel after the nucleic acid and the ionized lipids are completely bound in the first mixed solution before the second oil phase channel forms a laminar flow and mixes with the first mixed solution.

[0080] Specifically, when the aqueous phase solution and the first oil phase solution are crossed using the first and second channels to form a laminar flow, then allowed to flow through the stirring channel and then passed through the mixing module, it was confirmed that when the solution passes through the stirring section in the third mixing module, the nucleic acid in the aqueous phase solution and the ionized lipid in the first oil phase solution are mixed, exhibiting a mixing rate of 80% or more.

[0081] Specifically, the flow rate ratio of the aqueous phase solution injected into the first channel and the first oil phase solution injected into the second channel is 1:1 to 10:1, and may be 3:1 to 9:1. When mixed and used within the above range, lipid nanoparticles with a uniform particle size can be produced. More specifically, if it is contained below the above range, there is a problem that the ethanol content is high during the subsequent process, making it difficult to maintain the shape of lipid nanoparticles, and if it is contained above the above range, there is a problem that the movement of lipid particles in the aqueous phase solution is restricted due to the inclusion of a large amount of aqueous phase solution, resulting in the formation of particles with an excessively small diameter.

[0082] Thus, in the present invention, the binding position of the third channel with the stirring channel is determined taking into consideration the degree of binding between the nucleic acid and the ionized lipid, and is bound to the non-stirring section in the first mixing module, the non-stirring section in the second mixing module, the non-stirring section in the third mixing module, the non-stirring section in the fourth mixing module, or the non-stirring section in the fifth mixing module, so that the second oil phase solution forms a laminar flow with the first mixed solution, which then passes through the stirring module and is mixed with the first mixed solution to form a second mixed solution, and in the second mixed solution, the particles to which the nucleic acid and the ionized lipids are bound are bound to the non-ionized lipids, neutral lipids, and fusogenic lipids to form lipid nanoparticles containing nucleic acid.

[0083] Unlike the conventional method of producing lipid nanoparticles by mixing nucleic acid, ionized lipids, non-ionized lipids, neutral lipids and fusogenic lipids all at once, the present invention allows for the production of more uniform lipid nanoparticles by inducing binding in stages to form lipid nanoparticles.

[0084] The lipid nanoparticles prepared according to the present invention may be uniformly spherical and have a polydispersity index of 0.2 or less, 0.01 to 0.2, 0.05 to 02, or 0.1 to 0.2. Lipid nanoparticles satisfying the polydispersity index within the above range are provided as lipid nanoparticles having a very uniform size. As described above, when lipid nanoparticles having a uniform size are prepared, a separate operation for classifying lipid nanoparticles having a specific size is not required, so that the production yield can be maximized in the subsequent process after the mixing process for providing the lipid nanoparticles as a vaccine or therapeutic agent.

[0085] In addition, the lipid nanoparticles containing the nucleic acid of the present invention may contain the ionized lipid at 10 to 30 mol% based on the total weight of the lipid in the lipid nanoparticle. The ionized lipid may be contained at 15 to 19.9 mol% based on the total weight of the lipid in the lipid nanoparticle. As described above, even when the ionized lipid is contained at 20 mol% or more, it is possible to produce lipid nanoparticles having a uniform diameter compared to the conventional lipid nanoparticle manufacturing method, and the content range of the ionized lipid is not only the range of the prior patents discussed above, but also the commercialized mRNA vaccines, even when the content is low, the lipid nanoparticles produced can be produced to have a uniform size. That is, the content of the ionized lipid, which has a toxicity issue, is reduced to solve the toxicity problem, and in the manufacturing process, the ionized lipid is separated from other lipids and bound to the nucleic acid, thereby producing lipid nanoparticles having a uniform size and increasing the production yield.

[0086] The weight ratio of the ionizable lipid and the nucleic acid may be 3:1 to 50:1, 3.3:1 to 50:1, or 3.3:1 to 16.7:1. The molar ratio of the ionizable lipid, the non-ionizable lipid, the fusogenic lipid, and the neutral lipid may be within the range of 10 to 50:10 to 50:30 to 65:1 to 2.5. It can be confirmed that the content range of the fusogenic lipid is increased and the content of the ionizable lipid is decreased compared to the conventional technology.

[0087] As described above, in conventional manufacturing methods, when the content of ionized lipids is reduced, the size of the lipid nanoparticles produced is not uniform, and it is known that the content range of lipids, etc. cannot be adjusted in practice.

[0088] However, in the present invention, unlike conventional manufacturing methods, lipids are classified into ionized lipids and the remaining lipids, which are dissolved in various organic solvents, and then gradually combined with nucleic acids, thereby making it possible to produce lipid nanoparticles of uniform size while reducing the content of ionized lipids.

[0089] Lipid nanoparticles containing a low concentration of ionized lipid according to another embodiment of the present invention may be prepared by the above-mentioned preparation method.

[0090] As described above, the lipid nanoparticles produced by the production method of the present invention contain a low content of ionized lipids, which not only eliminates the toxicity problem caused by containing a large amount of ionized lipids, but also has a uniform particle size, which increases the production yield.

[0091] Another embodiment of the present invention relates to an apparatus for manufacturing lipid nanoparticles containing a low concentration of ionized lipids, the apparatus comprising: a first channel for flowing an aqueous phase solution containing nucleic acid; a second channel for flowing a first oil phase solution containing ionizable lipids; a third channel for flowing a second oil phase solution containing non-ionizable lipids, neutral lipids and fusogenic lipids; and a stirring channel, wherein the first channel and the second channel form a first intersection, the intersection being connected to the stirring channel, and the stirring channel may comprise a mixing module of a stirring section and a non-stirring section.

[0092] The mixing module is formed in a plurality of mixing modules in the stirring channel, and the n-th mixing module is formed in order based on the fluid flow direction of the stirring channel, and the n-th mixing module refers to the order of the mixing modules repeatedly formed in the stirring channel.

[0093] The stirring channel may include 3 to 70 mixing modules. The mixing module includes a stirring section and a non-stirring section as described above, and allows the aqueous phase solution and the oil phase solution flowing as laminar flows to be mixed in the stirring section. By repeating the stirring section and the non-stirring section multiple times in the stirring channel, the mixing efficiency can be increased.

[0094] The mixing modules in the stirring channel may be 3 to 70, 3 to 50, 3 to 40, 3 to 35, and preferably 30. When the mixing modules are included within the above range, as described above, the nucleic acid in the aqueous phase solution and the ionized lipid in the first oil phase solution are completely combined, and then the non-ionizable lipid, neutral lipid, and fusogenic lipid in the second oil phase solution are gradually combined to prepare lipid nanoparticles having a uniform size.

[0095] The third channel may be coupled to the stirring channel, forming an intersection between the first mixing module to the fifth mixing module.

[0096] The third channel is for flowing the second oil phase solution to form a laminar flow with the first mixed solution flowing in the stirring channel, and as explained above, the lipids, etc. in the second oil phase solution flowing in the third channel are bound to the stirring channel at a certain distance from the intersection of the first and second channels so that they can bind to the particles after the nucleic acids and ionized lipids are thoroughly mixed to form particles.

[0097] The connecting position of the third channel is between the first mixing module and the fifth mixing module, specifically, it can be connected to the non-stirring portion in the first mixing module, the non-stirring portion in the second mixing module, the non-stirring portion in the third mixing module, the non-stirring portion in the fourth mixing module, or the non-stirring portion in the fifth mixing module, preferably, it can be connected to the non-stirring portion in the second mixing module, the non-stirring portion in the third mixing module, the non-stirring portion in the fourth mixing module, or the non-stirring portion in the fifth mixing module, and more preferably, it can be connected to the non-stirring portion in the third mixing module.

[0098] As described above, by adjusting the binding position, the first mixed solution can be mixed through the stirring section of the first mixing module, the stirring section of the second mixing module, and the stirring section of the third mixing module before the second oil phase solution in the third channel is crossed with the first mixed solution, providing sufficient time for binding between the nucleic acid in the aqueous phase solution and the ionized lipids in the first oil phase solution.

[0099] The length of the mixing module may be 1 to 5 mm based on the flow direction of the fluid in the stirring channel. The length ratio of the stirring section and the non-stirring section may be 45:1 to 5:0.3 based on the flow direction of the fluid in the stirring channel. Specifically, the length of the stirring section may be 2 to 4 mm, the non-stirring section may be 0.1 to 0.25 mm, the length of the stirring section may be 2 to 3.5 mm, the non-stirring section may be 0.1 to 0.20 mm, the length of the stirring section may be 2 to 3 mm, and the non-stirring section may be 0.12 to 0.20 mm. By configuring the stirring section and the non-stirring section within the above ranges, binding between nucleic acids and lipids, etc. can be easily generated in the first mixed solution and the second mixed solution passing through the stirring section and the non-stirring section.

[0100] The stirring section has grooves formed therein to mix the inflowing fluids, and the grooves of the stirring section can generate chaotic mixing to increase the mixing efficiency of laminar flow in the stirring channel.

[0101] The shape of the groove may be a rectangle, a semicircle, or a triangle. The shape of the groove is for increasing the mixing efficiency of the fluid passing through the mixing channel, and is not limited to the above examples, and any groove shape that can increase the mixing efficiency may be used.

[0102] Specifically, Fig. 1 relates to a manufacturing apparatus for producing lipid nanoparticles of the present invention. Specifically, the manufacturing apparatus for producing lipid nanoparticles of the present invention includes a first channel 100, a second channel 200, a mixing channel 300, and a third channel 400, and the mixing module 310 in the mixing channel 300 is repeatedly formed.

[0103] The mixing module 310 is formed with a stirring section 311 and a non-stirring section 312 .

[0104] FIG. 2 relates to a conventional lipid nanoparticle manufacturing apparatus, and unlike FIG. 1, does not include a third channel 400' in the stirring channel 300'.

[0105] The manufacturing apparatus may be formed on a material selected from the group consisting of a glass substrate, a silicon wafer, or a polymer film, but the examples of the material are not limited to the above examples, and any material capable of forming a microchannel can be used.

[0106] The polymer film may be selected from the group consisting of polyimide, polyethylene, fluorinated ethylene propylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polysulfone, and mixtures thereof, but is not limited to the above examples.

[0107] For example, aluminum is deposited on a silicon wafer using an e-beam evaporator, and photoresist is patterned on the aluminum using photolithography. Then, aluminum is etched using the photoresist as a mask, and after the photoresist is removed, silicon is etched by DRIE (deep ion reactive etching) using the aluminum as a mask. After removing the aluminum, glass is bipolar-bonded on the wafer and sealed to manufacture the device.

[0108] The first, second, third and stirring channels in the manufacturing apparatus have an average diameter of 180 to 220 μm, preferably 200 μm, a height of 60 to 100 μm, preferably 80 μm, and a groove height of 10 to 50 μm, preferably 30 μm, but any channel diameter, height and groove height capable of producing lipid nanoparticles by the flow of solution can be used without any restrictions.

[0109] The flow rates of the aqueous phase solution, the first oil phase solution, and the second oil phase solution injected into the channel were 0.3 to 0.9 ml / min, 0.05 ml / min to 0.3 ml / min, and 0.05 ml / min to 0.3 ml / min, respectively. When the aqueous phase solution, the first oil phase solution, and the second oil phase solution are injected within the above ranges, lipid nanoparticles having a uniform size can be produced.

[0110] In addition, as described above, the nucleic acid and the ionized lipid must be sufficiently bound in the first mixed solution before the second oil phase solution crosses and mixes with the first mixed solution. In order to increase the binding index rate between the nucleic acid and the ionized lipid, the flow rates of the aqueous phase solution and the first oil phase solution injected into the channel must be adjusted, and the flow rate ratio may be 1:1 to 10:1, preferably 2:1 to 10:1, and more preferably 3:1 to 9:1. Within the above flow rate ratio range, the mixing index rate of the nucleic acid and the ionized lipid in the first mixed solution that has passed through the stirrer in the third mixing module may be 80% or more, and may be 85%. As described above, when the mixing index value is satisfied, it means that the nucleic acid and the ionized lipid are sufficiently bound, and thereafter, they can be bound to other lipids through the third channel to produce lipid nanoparticles.

[0111] Manufacturing Example 1 Manufacturing equipment for lipid nanoparticles Negative photoresist was applied to the surface of a silicon wafer by spin coating, then heated at 65℃ for 0-3 minutes and 95℃ for 6-9 minutes to evaporate the solvent, remove the groove structure, and irradiate with UV light.Then, the photoresist that was exposed to UV light was solidified by heating at 65℃ for 1-2 minutes and 95℃ for 6-7 minutes.

[0112] To form grooves in the stirring channels, negative photoresist was coated again by spinning, then heated at 65°C for 0-3 minutes and 95°C for 6-9 minutes to evaporate the solvent, and then irradiated UV light onto the chip's grooves. The photoresist was then heated at 65°C for 1-2 minutes and 95°C for 6-7 minutes to solidify the parts of the photoresist exposed to UV light. The parts not exposed to UV light were then removed using a developer.

[0113] The manufacturing equipment that was fabricated is as shown in Figure 3.

[0114] Manufacturing Example 2 Production of lipid nanoparticles An aqueous phase solution was prepared by mixing mRNA (CleanCap® Firefly Luciferase mRNA, ∼1,929 nucleotides) with 10 mM citrate solution (pH 3).

[0115] ALC-0315 was dissolved in ethanol as an ionizable lipid to prepare the first oil phase solution, followed by dissolving DSPC, cholesterol, and DMG-PEG2000 in ethanol to prepare the second oil phase solution.

[0116] In the production apparatus produced in Production Example 1, the aqueous phase solution was injected into the first channel, the first oil phase solution was injected into the second channel, and the second oil phase solution was injected into the third channel.

[0117] The aqueous phase solution was injected at a flow rate of 0.6 mL / min, the first oil phase solution at 0.1 mL / min, and the second oil phase solution at 0.1 mL / min.

[0118] After mixing was completed, the solution discharged from the device was passed through a dialysis (PES membrane dialysis cassette (MWCO = 10,000 daltons)) to exchange the buffer solution for PBS to produce lipid nanoparticles.

[0119] The contents of the components relative to the lipid nanoparticles are as shown in Table 1 below.

[0120] [Table 1]

[0121] The comparative example was produced in the same manner as the production example, except that a production apparatus without a third channel as shown in FIG. 2 was used, and ALC-0315, DSPC, cholesterol and DMG-PEG2000 were dissolved in ethanol and injected into the second channel as an oil phase solution.

[0122] In Example 3 and Comparative Example 2, the mRNA was produced in the same manner as in Production Example 2, except that CleanCap (registered trademark) Enhanced Green Fluorescent Protein mRNA (996 nucleotides) was used.

[0123] Experimental Example 1 Particle size distribution evaluation The particle size distribution (polydispersity index) of the lipid nanoparticles prepared in Preparation Example 2 and Comparative Example 1 was measured using a dynamic laser scattering device (Malvern Zetasizer).

[0124] The experimental results are shown in Figure 4.

[0125] Specifically, when containing 16.5 mol% ionized lipid, the PDI of the lipid nanoparticles produced using the manufacturing apparatus of the comparative example was 0.25±0.08, while the PDI of the lipid nanoparticles produced using the manufacturing apparatus of the manufacturing example was 0.15±0.01, confirming that lipid nanoparticles of uniform size were produced, as shown in Figure 5.

[0126] Moreover, in Example 2, the content of ionized lipids was increased and the product was produced using the production apparatus of the present invention, and it was confirmed that the PDI value was 0.08±0.03, which is 0.2 or less.

[0127] Example 3 and Comparative Example 2 were produced in the same manner using different mRNA. It was confirmed that the PDI of Example 3 was 0.15 and that of Comparative Example 2 was 0.24, indicating a difference in the uniformity of the diameter of the produced particles.

[0128] Experimental Example 2 SHM Mixing Evaluation An alternative experiment was carried out to confirm the degree of stirring between the aqueous phase solution and the first oil phase solution. Through this experiment, binding between the mRNA in the aqueous phase solution and the ionized lipid in the first oil phase solution was confirmed.

[0129] Specifically, instead of the aqueous phase solution, an aqueous phase solution was prepared by mixing 0.015 w / w% Rhodamine B in DI Water, and instead of the first oil phase solution, an oil phase solution was prepared by mixing 0.015 w / w% Rhodamine B in ethanol.

[0130] The aqueous phase solution and the oil phase solution were injected into the first and second channels, respectively. The flow rates and flow ratios at the time of injection into each channel were as follows:

[0131] [Table 2]

[0132] The mixed solution was injected into the first and second channels, and then the mixing index rate (%) was measured according to the difference in the flow rate ratio.

[0133] The following index image analysis method was used. 1. Convert the captured image file to Grey Scale using Image J. 2 Convert the image converted to Gray Scale to 8Bit 3. Background Intensity Filtering 4 Analyze the intensity and calculate the index for each pixel using the following formula:

[0134]

number

[0135] The experimental results are shown in Figure 6.

[0136] When the aqueous phase solution and the oil phase solution were mixed at a flow rate ratio of 1:1, they passed through the mixing module three times and showed a mixing ratio index of 82%. However, when mixed at a 1:1 ratio, the degree of mixing was relatively low, which means that when this is actually used as the aqueous phase solution and the oil phase solution for producing lipid nanoparticles, the mRNA in the aqueous phase solution may not bind sufficiently to the ionized lipids in the first oil phase solution.

[0137] As a result, in some experiments, when the aqueous phase solution and the oil phase solution are mixed at a flow rate ratio of 3:1 to 9:1, the solutions pass through the third mixing module and are mixed to a high degree of 87% and 95%, respectively. Through the experiments, it can be confirmed that the preferred flow rate ratio for mixing the aqueous phase solution and the first oil phase solution is 3:1 to 9:1.

[0138] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also belong to the scope of the present invention. [Industrial Applicability]

[0139] The present invention relates to a method and an apparatus for producing lipid nanoparticles (LNPs). [Explanation of symbols]

[0140] 100 Channel 1 100' Channel 1 200 Channel 2 200' Channel 2 300 mixing channels 300' Mixing Channel 310 Mixed Module 310' Mixed Module 311 Mixing section 311' Mixing section 312 Non-mixing section 312' Non-mixing section 400 Channel 3 500 leaked channels 500' Outflow Channel

Claims

1. Producing an aqueous phase solution containing nucleic acid; Dissolving a positively charged ionizable lipid in an organic solution to prepare a first oil phase solution; dissolving a non-ionizable lipid, a neutral lipid and a fusogenic lipid in an organic solution to prepare a second oil phase solution; injecting and flowing the aqueous phase solution into a first channel; Injecting and flowing the first oil phase solution into a second channel; the aqueous phase solution and the first oil phase solution cross each other and flow through a stirring channel as a first mixed solution; A step of flowing the second oil phase solution into a third channel connected to the stirring channel to cross the first mixed solution and mix it into a second mixed solution; and passing the second mixed solution through a plurality of stirred and unstirred portions in the stirring channel to form lipid nanoparticles containing nucleic acids; said neutral lipid is selected from the group consisting of polyethylene glycol 2000 distearoylphosphatidylethanolamine (PEG(2000)DSPE), DMG-PEG, PEG-DMPE, DPPE-PEG, DPG-PEG, PEG-DOPE, and mixtures thereof; A method for producing lipid nanoparticles, wherein a flow rate ratio of the aqueous phase solution injected into the first channel and the first oil phase solution injected into the second channel is 1:1 to 9:

1.

2. The method for producing lipid nanoparticles described in claim 1, wherein the stirring channel includes a mixing module of a stirring section and a non-stirring section.

3. The mixing module is formed in plurality within the stirring channel, An n-th mixing module is formed in sequence based on the flow direction of the fluid in the mixing channel, The method for producing lipid nanoparticles according to claim 2 , wherein the nth mixing module means the order of mixing modules repeatedly formed in the stirring channel.

4. The method for producing lipid nanoparticles according to claim 3, wherein the third channel forms an intersection between the first mixing module to the fifth mixing module and is connected to the stirring channel, so that the first mixed solution and the second oil phase solution intersect to form a laminar flow.

5. The method for producing lipid nanoparticles according to claim 1, wherein the lipid nanoparticles are uniformly spherical and have a polydispersity index of 0.2 or less.

6. The method for producing lipid nanoparticles according to claim 1 , wherein the ionized lipid is contained in an amount of 10 to 30 mol % based on the total weight of lipid in the lipid nanoparticles.

7. The method for producing lipid nanoparticles according to claim 1, wherein the weight ratio of the ionizable lipid to the nucleic acid is from 3:1 to 50:

1.

8. The method for producing lipid nanoparticles according to claim 1, wherein the nucleic acid is selected from the group consisting of RNA, DNA, siRNA (short interfering RNA), mRNA (messenger RNA), aptamer, antisense ODN (antisense oligodeoxynucleotide), antisense RNA, ribozyme, DNAzyme, and mixtures thereof.

9. The method for producing lipid nanoparticles according to claim 1, wherein the non-ionized lipid is selected from the group consisting of DSPC (distearoylphosphatidylcholine), DOPE (dioleolphosphatidyl ethanolamine), DPPE (bis(diphenylphosphino) ethanol), diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylserine, and mixtures thereof.

10. The method for producing lipid nanoparticles according to claim 1, wherein the fusogenic lipid is selected from the group consisting of phospholipids, cholesterol, tocopherol and mixtures thereof.

11. A first channel through which an aqueous phase solution containing nucleic acids flows; A second channel for flowing a first oil phase solution containing an ionizable lipid; a third channel for flowing a second oil phase solution containing a non-ionizable lipid, a neutral lipid and a fusogenic lipid; a stirring channel; the first channel and the second channel form a first intersection, the first intersection being connected to the stirring channel; The stirring channel includes a stirring section and a non-stirring section mixing module; The mixing module is formed in a plurality of mixing modules in the stirring channel, n-th mixing modules are formed in sequence based on the flow direction of the fluid in the mixing channel, The nth mixing module means the order of mixing modules repeatedly formed in the mixing channel; The third channel is connected to the stirring channel by forming an intersection between the first mixing module to the fifth mixing module of the mixing module, The apparatus for producing lipid nanoparticles, wherein a flow rate ratio of the aqueous phase solution injected into the first channel and the first oil phase solution injected into the second channel is 3:1 to 9:

1.

12. The lipid nanoparticle manufacturing apparatus of claim 11, comprising 3 to 70 mixing modules within the stirring channel.

13. The lipid nanoparticle manufacturing apparatus according to claim 11 , wherein the length of the mixing module is 1 to 5 mm based on the flow direction of the fluid in the stirring channel.

14. The lipid nanoparticle manufacturing apparatus according to claim 11 , wherein the length ratio of the stirring section and the non-stirring section is 45:1 to 5:0.3 based on the flow direction of the fluid in the stirring channel.

15. The lipid nanoparticle manufacturing apparatus according to claim 11 , wherein the stirring unit has a groove formed therein for mixing the inflowing fluid.

16. The lipid nanoparticle manufacturing apparatus according to claim 15, wherein the grooves of the mixing section generate chaotic mixing to enhance the mixing efficiency of laminar flow in the mixing channel.

17. The apparatus for producing lipid nanoparticles described in claim 15, wherein the shape of the groove is a square, semicircle or triangle.

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