Lipid nanoparticle manufacturing chip, lipid nanoparticle manufacturing system including the same, and lipid nanoparticle manufacturing method

Abstract

The chip for producing lipid nanoparticles includes a first raw material supply flow path, a second raw material supply flow path, and a mixer section connected to the first raw material supply flow path and the second raw material supply flow path, and mixes the first raw material supplied through the first raw material supply flow path and the second raw material supplied through the second raw material supply flow path. The mixer section includes a first stabilization section, and a first mixing section connected to the first stabilization section, in which the first raw material and the second raw material are mixed with each other. The mixing of the first raw material and the second raw material is performed more in the first mixing section than in the first stabilization section.

Description

[Technical Field]

[0001] The present invention relates to a lipid nanoparticle production chip, a lipid nanoparticle production system including the lipid nanoparticle production chip, and a lipid nanoparticle production method, and more particularly to a lipid nanoparticle production chip for producing lipid nanoparticles containing an active ingredient such as mRNA, a lipid nanoparticle production system including the lipid nanoparticle production chip, and a lipid nanoparticle production method containing the active ingredient. [Background technology]

[0002] Messenger RNA (mRNA) is a precursor to protein synthesis and contains genetic information. It provides access to various therapeutic agents and can be used as preventive or therapeutic vaccines. Deficient proteins can also be synthesized through mRNA. The advantage of mRNA therapeutics is that, unlike DNA, they do not need to be transported to the nucleus and are not inserted into a dielectric medium. This prevents permanent genetic disorders and provides a high level of safety. Furthermore, mRNA can synthesize deficient proteins within cells that are inaccessible to protein therapeutics. mRNA can vary in size depending on the protein being expressed and exists as a single strand. mRNA produced from DNA escapes from the nucleus into the cytoplasm, where it interacts with ribosomes to produce proteins. While mRNA has been gaining attention as a next-generation gene therapy, its single-stranded nature makes it highly unstable. It is rapidly degraded by nucleases in the blood and excreted quickly through the kidneys. It also carries a strong negative charge, making it difficult to pass through cell membranes.

[0003] Safe and effective drug delivery technologies for treatment using anionic drugs, including nucleic acids, have been studied for a long time, and various delivery vehicles and delivery technologies have been developed. Extensive research has been conducted on mRNA vaccines, which use a method of delivering mRNA by encapsulating it in lipid nanoparticles, and research into mass production systems for this vaccine is ongoing.

[0004] Recently, the method of delivering mRNA by encapsulating it in lipid nanoparticles has been gaining attention in the fields of pharmaceuticals, vaccines, and drug delivery systems (DDS), but mass production has been difficult. There is a bulk production method in which turbulence is generated in a large container to mix, dilute, and concentrate, but this method has problems such as difficulty in obtaining uniform quality in the produced lipid nanoparticles and difficulty in controlling the production process.

[0005] Meanwhile, in an effort to solve these problems, methods for producing nanolipid particles using a chip-type mixer have been studied, but due to the difficulty in analyzing the characteristics of the fluid flow, it is difficult to produce nanolipid particles more efficiently and with higher quality. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US 10,835,878 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the technical problem of the present invention is to address this issue, and an object of the present invention is to provide a chip for producing lipid nanoparticles.

[0008] Another object of the present invention is to provide a lipid nanoparticle production system including the chip for producing lipid nanoparticles.

[0009] It is yet another object of the present invention to provide a method for producing lipid nanoparticles. [Means for solving the problem]

[0010] According to one embodiment of the present invention, a chip for producing lipid nanoparticles includes a first raw material supply channel, a second raw material supply channel, and a mixer unit connected to the first raw material supply channel and the second raw material supply channel and configured to mix a first raw material supplied through the first raw material supply channel with a second raw material supplied through the second raw material supply channel. The mixer unit includes a first stabilization unit and a first mixing unit connected to the first stabilization unit and configured to mix the first raw material and the second raw material with each other. The mixing of the first raw material and the second raw material is performed more frequently in the first mixing unit than in the first stabilization unit.

[0011] In one embodiment of the present invention, the first stabilizing portion may include a left stabilizing channel having a first width and a first length and a right stabilizing channel having the first width and the first length.

[0012] In one embodiment of the present invention, the left and right stabilizing channels of the first stabilizing portion may be symmetrical to each other and have an elliptical or circular shape.

[0013] In one embodiment of the present invention, the first mixing section includes a left mixing channel having a second width and a right mixing channel having a third width, and the second width and the third width may be different from each other.

[0014] In one embodiment of the present invention, the left and right mixing channels of the first mixing part may be elliptical or circular.

[0015] In one embodiment of the present invention, the first stabilizing unit may be connected to the first raw material supply line and the second raw material supply line. The first raw material flows through the first raw material supply line, the first stabilizing unit, and the first mixing unit in this order, and the second raw material flows through the second raw material supply line, the first stabilizing unit, and the first mixing unit in this order. The first raw material and the second raw material are mixed at an interface between the first raw material and the second raw material, and the first raw material may be mixed more in the first mixing unit than in the first stabilizing unit.

[0016] In one embodiment of the present invention, the first mixing section may be connected to the first raw material supply passage and the second raw material supply passage, the first raw material may flow through the first raw material supply passage, the first mixing section, and the first stabilizing section in that order, and the second raw material may flow through the second raw material supply passage, the first mixing section, and the first stabilizing section in that order.

[0017] In one embodiment of the present invention, the mixer unit may further include a second stabilizing unit connected to the first mixing unit, and a second mixing unit connected to the second stabilizing unit.

[0018] According to one embodiment of the present invention, a lipid nanoparticle production system includes a lipid nanoparticle production chip including a first raw material supply unit for supplying a first raw material, a second raw material supply unit for supplying a second raw material, a mixer unit for mixing the first raw material and the second raw material to form a mixed solution, and a lipid nanoparticle acquisition unit for obtaining lipid nanoparticles produced by the lipid nanoparticle production chip. The mixer unit of the lipid nanoparticle production chip includes a first stabilization unit and a first mixing unit connected to the first stabilization unit, where the first raw material and the second raw material are mixed with each other.

[0019] In one embodiment of the present invention, the first stabilizing section may include a left stabilizing channel having a first width and a first length and a right stabilizing channel having the first width and the first length. The first mixing section may include a left mixing channel having a second width and a right mixing channel having a third width, and the second width and the third width may be different from each other.

[0020] According to one embodiment of the present invention, a method for producing lipid nanoparticles includes the steps of preparing a first raw material containing an active ingredient and a second raw material containing a lipid, mixing the first raw material with the second raw material to form lipid nanoparticles containing the active ingredient, and filtering and filling a solution containing the lipid nanoparticles into individual containers to produce a final product. The step of forming lipid nanoparticles is performed on a chip for producing lipid nanoparticles having channels formed therein, and includes a stabilization step in which the first raw material and the second raw material pass through a stabilization section including a left stabilization channel and a right stabilization channel having the same width and length, and a mixing step in which the first raw material and the second raw material pass through a mixing section including a left mixing channel and a right mixing channel having different widths to mix with each other.

[0021] In one embodiment of the present invention, the left and right stabilizing channels of the stabilizing unit may be symmetrically elliptical or circular, and the left and right mixing channels of the mixing unit may be elliptical or circular.

[0022] In one embodiment of the present invention, the stabilizing step and the mixing step may be alternately repeated at least two times. [Effects of the Invention]

[0023] The mixer unit of the chip for producing lipid nanoparticles according to the embodiment of the present invention includes a stabilizing unit and a mixing unit arranged alternately. The flow path design of the stabilizing unit and the mixing unit allows for more stable and efficient production of high-quality lipid nanoparticles.

[0024] However, the effects of the present invention are not limited to the above effects, and may be variously expanded within the scope of the present invention. [Brief explanation of the drawings]

[0025] [Figure 1] 1 is a schematic diagram of a lipid nanoparticle production system according to one embodiment of the present invention. [Figure 2] FIG. 2 is a diagram showing in detail the mixer section of the lipid nanoparticle production chip of the lipid nanoparticle production system of FIG. 1. [Figure 3] FIG. 3 is a diagram for explaining the flow of fluid in the mixer section of FIG. 2. [Figure 4] FIG. 2 is a diagram showing in detail the mixer section of the lipid nanoparticle producing chip of the lipid nanoparticle producing system according to one embodiment of the present invention. [Figure 5] FIG. 1 is a diagram showing a chip for producing lipid nanoparticles in a lipid nanoparticle producing system according to one embodiment of the present invention. [Figure 6] FIG. 10 is a diagram showing the results of simulation and experiment of the fluid flow in the chip for producing lipid nanoparticles in the lipid nanoparticle producing system according to one embodiment of the present invention. [Figure 7] FIG. 7 is a diagram for explaining the calculation of the mixing index for the experiment of FIG. 6. [Figure 8] 10 is a graph showing the results of a simulation and experiment on the degree of mixing depending on the length of a mixer unit of a chip for preparing lipid nanoparticles in a system for preparing lipid nanoparticles according to an embodiment of the present invention. [Figure 9] 1 is a flowchart illustrating a method for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 10] 10 is a flowchart showing in detail the steps of producing lipid nanoparticles on a chip in the method of producing lipid nanoparticles of FIG. 9. DETAILED DESCRIPTION OF THE INVENTION

[0026] Preferred embodiments of the present invention will now be described in more detail with reference to the drawings.

[0027] Since the present invention can be modified in various ways and can have various forms, specific embodiments are shown in the drawings and described in detail herein, but it is not intended to limit the present invention to the specific disclosed forms, and it should be understood that the present invention includes all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention.

[0028] FIG. 1 is a schematic diagram of a lipid nanoparticle production system according to one embodiment of the present invention.

[0029] Referring to FIG. 1, the lipid nanoparticle production system includes a first raw material supply unit 10 containing mRNA, a second raw material supply unit 20 containing lipids, a lipid nanoparticle acquisition unit 30, and a chip 100 for producing lipid nanoparticles.

[0030] The first raw material supply unit 10 stores a first raw material and provides the first raw material to the lipid nanoparticle manufacturing chip 100. The first raw material may be a solution containing mRNA. For example, the first raw material may include, but is not limited to, mRNA and water. For example, the first raw material may include various forms of active ingredients, such as nucleic acids such as miRNA, siRNA, and DNA, or CRISPR, in addition to mRNA.

[0031] The second raw material supply unit 20 stores a second raw material and provides the second raw material to the lipid nanoparticle manufacturing chip 100. The second raw material may be a solution containing lipid. For example, the second raw material may contain lipid and ethanol.

[0032] The lipid nanoparticle producing chip 100 may produce a lipid nanoparticle solution containing mRNA by mixing the first raw material and the second raw material.

[0033] The lipid nanoparticle producing chip 100 may include a first raw material supply channel 110, a second raw material supply channel 120, a mixer unit 200, and a lipid nanoparticle obtaining channel .

[0034] The first raw material supply passage 110 may receive the first raw material from the first raw material supply unit 10 and transfer it to the mixer unit 200. The second raw material supply passage 120 may receive the second raw material from the second raw material supply unit 20 and transfer it to the mixer unit 200.

[0035] The mixer unit 200 may be connected to the first and second raw material supply channels 110 and 120 and may mix the first raw material and the second raw material to prepare a mixture containing lipid nanoparticles. The mixer unit 110 may be a microfluidic mixer commonly used in microchannels, such as a chaotic mixer or a herringbone mixer. In this case, a mixture containing lipid nanoparticles formed by self-assembly of lipids and mRNA at the interface between the two fluids may be prepared by mixing the first raw material and the second raw material in the channel.

[0036] The mixer unit 200 may include at least one pair of stabilizing and mixing units arranged alternately. To form high-quality lipid nanoparticles containing the active ingredient of the first raw material by surrounding the active ingredient with the lipid of the second raw material, optimal design is required, as not only the mixing between the solutions (first and second raw materials) but also the area, shape, and flow of the contact surface between the solutions affect the formation of the active ingredient. In an embodiment of the present invention, this is achieved by configuring the mixer unit 200 to include at least one pair of stabilizing and mixing units, as described above. The mixer unit 200 will be described in detail below in the description of FIG. 2.

[0037] The lipid nanoparticle acquisition channel 130 is connected to the mixer unit 200, and the lipid nanoparticle acquisition unit 30 can obtain a mixture containing the lipid nanoparticles formed in the mixer unit through the lipid nanoparticle acquisition channel 130.

[0038] The mixture may then be diluted or concentrated as needed to form a lipid nanoparticle solution of the desired concentration, and the lipid nanoparticle-containing solution may then be filtered and filled into individual containers to produce the final product.

[0039] The chip 100 for producing lipid nanoparticles may have various structures for forming a channel therein, for example, a structure including a groove on a first substrate and a second substrate on the first substrate that is bonded to the first substrate.

[0040] FIG. 2 is a diagram showing in detail the mixer section of the lipid nanoparticle production chip of the lipid nanoparticle production system of FIG.

[0041] The mixer section 200 may include a combining section 202 , a first stabilizing section 210 , a first mixing section 220 , a second stabilizing section 230 , a second mixing section 240 , and a discharge section 204 .

[0042] The merging section 202 is a point where a first raw material supply passage (110 in FIG. 1) and a second raw material supply passage (120 in FIG. 1) merge. A first stabilizing section 210 may be connected to the merging section 202.

[0043] The first stabilizing section 210 may include a left stabilizing channel 212 having a first width w1 and a first length and a right stabilizing channel 214 having the first width w1 and the first length. Thus, the left stabilizing channel 212 and the right stabilizing channel 214 of the first stabilizing section 220 may be elliptical or circular and symmetrical to each other with respect to the second direction D2. In the merging section 202, the first raw material and the second raw material begin to mix, and lipid nanoparticles formed by self-assembly at the mixing interface may pass through the channels of the first stabilizing section 220 so that they have sufficient time to form a structure.

[0044] The first mixing unit 220 is connected to the first stabilizing unit 210. The first mixing unit 220 includes a left mixing channel 222 having a second width w2 and a right mixing channel 224 having a third width w3, where the second width w2 and the third width w3 are different from each other. Thus, the left mixing channel 222 and the right mixing channel 224 of the first mixing unit 220 may have an asymmetric elliptical or circular shape. Mixing of the first raw material and the second raw material may occur more frequently in the first mixing unit 220 than in the first stabilizing unit 210.

[0045] The second stabilizing unit 230 is connected to the first mixing unit 220. The second stabilizing unit 230 may include a left stabilizing channel 232 having the first width w1 and the first length and a right stabilizing channel 234 having the first width w1 and the first length. Thus, the left stabilizing channel 232 and the right stabilizing channel 234 of the second stabilizing unit 230 may be symmetrical to each other and have an elliptical or circular shape. Lipid nanoparticles formed by self-assembly at the fluid mixing interface in the first mixing unit 220 may pass through the channels of the second stabilizing unit 230 so that they have sufficient time to form a structure.

[0046] The second mixing unit 240 is connected to the second stabilizing unit 230. The second mixing unit 240 may include a left mixing channel 242 having the third width w3 and a right mixing channel 244 having the second width w2. Thus, the left mixing channel 242 and the right mixing channel 244 of the second mixing unit 240 may have an asymmetric elliptical or circular shape, or may have a symmetrical shape to the shape of the first mixing unit 220. Mixing of the first raw material and the second raw material may be performed more in the second mixing unit 240 than in the second stabilizing unit 230.

[0047] The discharge section 204 may be connected to the second mixing section 240 and to a lipid nanoparticle acquisition channel (130 in FIG. 1).

[0048] In this embodiment, the mixer unit 200 includes two pairs of stabilizing units and mixing units that are alternately arranged, but is not limited thereto. For example, various structures are possible, such as more stabilizing units and mixing units being alternately arranged, or the mixing unit being arranged before the stabilizing unit.

[0049] FIG. 3 is a diagram for explaining the flow of fluid in the mixer section of FIG.

[0050] Generally, nanolipid particles are produced through a self-assembly process in which particles are structured at the interface between two different solutions (water base, oil base) that occurs when the solutions are mixed. The main factors in designing a chip for producing nanolipid particles using microfluidic technology are a) the flow rate conditions of the applied solution, b) the Reynolds number, which quantifies the dynamic relationship of the relative forces due to the inertia and viscosity of the internal solution as a dimensionless number, and c) an analysis of the diffusion phenomenon that occurs due to the difference in concentration between the solutions at the interface and the momentum due to the flow of the solution fluid.

[0051] In addition, the flow rate conditions of the Flow Rate Ratio (FRR), which is the supply ratio of the water base and oil base solutions, and the Total Flow Rate (TFR) may be considered. The FRR is the ratio of each of the two solutions.

number

[0052] The dimensionless Reynolds number can be used to predict the degree of mixing and contact between solutions in the channel of a chip for producing nanolipid particles. The Reynolds number (Re) for the flow in the channel can be calculated using the following equation (1).

[0053]

number

[0054] ρ is the density of the inner solution, U mean is the average velocity of the solution, D h is the hydraulic diameter, which reflects the hydraulic characteristics of the diameter within the channel, and μ is the viscosity coefficient of the solution. Generally, when the Reynolds number is 2,300 or less, the flow of the solution can be defined as laminar flow, and when the Reynolds number is higher than 2,300, it passes through transition flow and finally becomes turbulent flow. As can be seen from the above variables, appropriately adjusting and tuning the Reynolds number, which is the dimensionless ratio of inertia and viscosity, has a positive effect on the self-assembly of nanolipid particles within the solution through the degree of advection of the internal solution and diffusion due to concentration differences at the interface and momentum diffusion, enabling the efficient production of high-quality nanolipid particles. Based on the above study, the channel of the chip for producing nano lipid particles was designed taking into consideration the influence of the degree of advection due to the Reynolds number under the flow rate condition, the influence of momentum diffusion, and the influence of diffusion due to concentration differences.

[0055] Meanwhile, the hydraulic diameter, which reflects the characteristics of the inner diameter of the channel, has an important effect on the adjustment and tuning of the Reynolds number, and the hydraulic diameter can be calculated by the following Equation 2, assuming that the channel is filled with the solution.

[0056]

number

[0057] Furthermore, the Reynolds number is closely related to the internal solution and fluid properties. Density and viscosity are particularly important factors to consider. The degree of mixing varies depending on the FRR, but the solution can essentially be considered a water-based and oil-based mixture. Since water and oil do not mix well, the viscosity of the mixture varies greatly depending on the FRR and the mixing ratio. When using ethanol and water, which are widely used in the production of nanolipid particles, the change in viscosity depending on the degree of mixing of water and ethanol can be seen in Table 1 below, based on the research of Khattab, I.

[0058] [Table 1]

[0059] At room temperature (298K), the viscosity (μ) of unmixed water with ethanol is calculated at 0.8914 mPa·s and measured at 0.8914 mPa·s, while that of pure ethanol is calculated at 1.099 mPa·s and measured at 1.099 mPa·s. However, due to the low degree of miscibility between water and ethanol, the viscosity of nano-sized particles generated during mixing is significantly different, with a calculated value of 2.161 mPa·s and measured value of 2.423 mPa·s for an ethanol mole fraction of 0.316. Therefore, only by carefully analyzing the FRR and the resulting degree of viscosity change and calculating the Reynolds number more accurately can we design optimized chip channels for nanolipid particle production.

[0060] Meanwhile, the diffusion phenomenon that occurs at the interface between two solutions can be analyzed in two ways. First, there is diffusion from a microscopic perspective due to the concentration difference that occurs between the solutions, and diffusion from a macroscopic momentum perspective due to the viscosity that occurs when mixing and the change in the fluid properties of the mixed solution. In the case of diffusion due to concentration difference, the relationship between the diffusion amount of the diffusing substance and the concentration difference can be quantified through several assumptions using Fick's Law, as shown in the following <Equation 3>, and reflected in the design.

[0061]

number

[0062] Analysis from a momentum perspective may be performed using velocity correlations to derive the two most commonly used models for fluid dynamics analysis: the mass conservation continuity equation and the Navier-Stokes equation (see Equation 4 below).

[0063]

number

[0064] Using these two methods, the performance of various channel designs for chips for producing nanolipid particles was analyzed through simulation and fluid dynamics analysis, and the results were verified through comparison with the simulation and analytical values through experiments.

[0065] In this embodiment of the present invention, the channel design is based on the operating principle of chaotic advection, which adds a chaotic effect that divides and breaks up the fluid flow to the phenomenon of laminar flow advection, which transports particles or substances in the main fluid flow direction. The chaotic advection method overcomes the biggest drawback of conventional advection, which is that transportation is restricted to the unidirectional flow of the main fluid, and can realize more efficient mixing between solutions.

[0066] In order to enhance the passive chaotic advection effect due to differences in geometric structures in microfluidic-based chips, it is possible to achieve high mixing efficiency even with low pressure changes by designing a) Dean vortexes that pass through channels in a chip with a circular structure, and b) channels that split, rearrange, and then recombine the fluid flow.

[0067] However, this method merely increases the mixing efficiency of two different solutions (water base, oil base) and is not fully applicable to the production of nanolipid particles. Further consideration must be given to the characteristics of nanolipid particle production, which involves self-assembly at the interface between the solutions during mixing. The applicant precisely tuned the channel design to maximize the self-assembly at the interface between the solutions during mixing and to produce nanolipid particles with high efficiency, and designed a chip for nanolipid particles including a stabilizing section and a mixing section according to an embodiment of the present invention. In addition to the general characteristics described above using chaotic advection, the chip was designed and engineered to apply symmetric and asymmetric structures to efficiently mix solution fluids and to more stably and efficiently produce high-quality nanolipid particles.

[0068] Referring to Figure 3, the circular symmetric and asymmetric structures are designed to increase the contact area between the two solutions through the Dean vortex effect when they pass through their respective channels, thereby enhancing not only mixing but also self-assembly of nanolipid particles. In particular, the strength of the Dean vortex can be defined as the dimensionless Dean number, as shown in Equation 5 below.

[0069]

number

[0070] The Dean Number is the relationship between the inertial force, centrifugal force, and viscous force applied to a fluid in a circular channel; ρ is the density of the internal solution; U_mean is the mean velocity of the solution; D_h is the hydrodynamic diameter; μ is the viscosity coefficient of the solution; and R_c is the radius of curvature. By specifically adjusting the channel width of the asymmetric section and the ratio between the wide section (see w2 in Figure 2) and the narrow section (see w3 in Figure 2), we were able to change the hydrodynamic diameter and radius of curvature between the asymmetric channels. This resulted in a) an increase in the strength and influence of Dean vortices in the asymmetric section, and b) a higher mixing efficiency in a specific section due to the increase in the area affected by Dean vortices, thereby forming an optimal contact area between solutions for the production of nanolipid particles. Furthermore, by applying a mixture of symmetric and asymmetric structures, the symmetric and asymmetric connecting section where the flow in the channel reunites increases and expands, resulting in an increase in the contact area between solutions and highly efficient mixing due to the asymmetric inertial collision of the channel and the formation of detailed vortices in various directions.

[0071] That is, in this embodiment, the fluid flow is split, collided with each other, and mixed, and then lipid nanoparticles are formed and stabilized through a self-assembly process after passing through a mixing stabilization reaction section, and the mixing and self-assembly process may be efficiently carried out through a Dean vortex.

[0072] In addition, the relatively low mixing efficiency in the symmetric section and the high mixing efficiency in the asymmetric section were designed to achieve sequential homogeneity mixing rather than immediate homogeneity mixing between the water and oil base solutions through a serial arrangement. The reason for this is that the initial lipid-based initial vesicles formed when phospholipids dissolved in the oil base solution meet the water base solution at the interface and assemble into disks due to their hydrophobic tails are more actively formed at a specific oil base solution concentration, e.g., the critical ethanol concentration in the case of ethanol, and this is thought to be closely affected by the residence time in the corresponding section during the mixing process. Therefore, the design allows for sequential dilution rather than abrupt dilution based on the concentration of the oil base solution, which increases the residence time of the mixed solution at the critical concentration, thereby increasing the efficiency of nanolipid particle production.

[0073] FIG. 4 is a diagram showing in detail a mixer unit of a chip for producing lipid nanoparticles in a system for producing lipid nanoparticles according to an embodiment of the present invention.

[0074] 4, the mixer unit 500 of the chip for producing lipid nanoparticles is substantially the same as the mixer unit of the chip for producing lipid nanoparticles in FIG. 2, except for the arrangement of the stabilizing unit and the mixing unit. Therefore, repeated description will be omitted.

[0075] The mixer section 500 may include a combining section 502 , a first mixing section 510 , a first stabilizing section 520 , a second mixing section 530 , a second stabilizing section 540 , and a discharge section 504 .

[0076] The merging section 502 is a point where the first raw material supplying passage and the second raw material supplying passage are merged. The merging section 502 may be connected to the first mixing section 510.

[0077] The first mixing section 510 includes a left mixing channel 512 having a third width w3 and a right mixing channel 514 having a second width w2, the second width w2 and the third width w3 being different from each other.

[0078] The first stabilizing section 520 is connected to the first mixing section 510. The first stabilizing section 520 may include a left stabilizing channel 522 having a first width w1 and a first length and a right stabilizing channel 524 having the first width w1 and the first length.

[0079] The second mixing section 530 is connected to the first stabilizing section 520. The second mixing section 530 may include a left mixing channel 532 having the second width w2 and a right mixing channel 534 having the third width w3.

[0080] The second stabilizing section 540 is connected to the second mixing section 530. The second stabilizing section 540 may include a left stabilizing channel 542 having the first width w1 and the first length and a right stabilizing channel 544 having the first width w1 and the first length.

[0081] The discharge section 204 may be connected to the second stabilizing section 540 and to a lipid nanoparticle acquisition channel.

[0082] FIG. 5 is a diagram showing a chip for producing lipid nanoparticles in a lipid nanoparticle producing system according to one embodiment of the present invention.

[0083] 5, the chip for producing lipid nanoparticles is substantially the same as the chip for producing lipid nanoparticles shown in FIG. 1, except that the mixer section is divided into a first mixer section 200a and a second mixer section 200b, and a connecting channel 140 connecting these sections is further formed. Therefore, repeated description will be omitted.

[0084] The lipid nanoparticle chip 100 may include a first raw material supply channel 110, a second raw material supply channel 120, a first mixer unit 200a, a connecting channel 140, a second mixer unit 200b, and a lipid nanoparticle acquisition channel 130.

[0085] The first mixer unit 200a and the second mixer unit 200b are connected by the connecting channel 140. The first mixer unit 200a and the second mixer unit 200b may each include a stabilizing unit and a mixing unit arranged alternately. Even if the number of the stabilizing units and mixing units is increased, the spatial design of the lipid nanoparticle chip can be made more efficient by arranging them in a plurality of rows or columns and connecting them using a connecting channel.

[0086] Figure 6 is a diagram showing the results of a simulation and experiment of the fluid flow in a chip for producing lipid nanoparticles in a system for producing lipid nanoparticles according to an embodiment of the present invention. Figure 7 is a diagram for explaining the calculation of the mixing index for the experiment in Figure 6. Figure 8 is a graph showing the results of a simulation and experiment of the degree of mixing depending on the length of the mixer unit in a chip for producing lipid nanoparticles in a system for producing lipid nanoparticles according to an embodiment of the present invention.

[0087] 6 and 8 show the simulation values of flow at four locations on the lipid nanoparticle production chip (see squares, four locations with different mixing lengths (lmix)), the corresponding actual experimental results, and the simulated values of fluid flow at the cross section of the channel (flow path) at the corresponding locations (bottom row). Similarity between the simulation and flow analysis values and the actual experimental values was confirmed, confirming the effectiveness of the optimal channel design in which stabilization sections and mixing sections are alternately arranged through the simulation and flow analysis.

[0088] The working fluids used in the experiment were deionized water as the water base solution and a mixed solution of rhodamine B diluted to a concentration of 50 μM in ethanol as the oil base solution. Images of the contact surface between the two solutions in the mixer were taken from a direction perpendicular to the top of the mixer. The intensity based on the pixels of the captured image was analyzed through n-by-m pixels by dividing the horizontal direction (x-axis) of the image by the number of n pixels and the vertical direction (y-axis) by the number of m pixels. Images were taken when the fluid flow at the measurement location, corresponding to each flow rate, stabilized in a steady state that did not change over time. For more accurate relative analysis, images were taken when the Rhodamine B and ethanol mixed solution was flowing and when only deionized water was flowing. When only the Rhodamine B and ethanol mixed solution was flowing, the value was reflected as 100% mixing, i.e., a state in which full mixing had been performed, and when only deionized water was flowing, the value was reflected as 0% mixing, a state in which no mixing had yet been performed. The images taken in the experiment were converted to grayscale, and the pixel intensity of each pixel was calculated. For each image taken, the pixel intensity and the intensity values at 100% mixing and 0% mixing were linearly interpolated to determine the standard deviation (σ), and the standard deviation (σ) of the initial section image in which mixing had not yet occurred within the micromixer was calculated. initial The mixing index was calculated and quantified based on the ratio of the total weight of the mixture to the total weight of the water (see Figure 7).

[0089] To verify the mixing index with the experimental results, COMSOL Multiphysics, a commonly used program for numerical analysis, was used for the analysis. The flow field distribution, which is a function of spatial and temporal coordinates, of the solution flow within the mixer when a flow rate is supplied was calculated by analyzing the Navier-Stokes equations shown above in Equation 4. The conditions applied to the calculation of the flow field distribution reflected three dimensions (x-, y-, and z-directions), a steady state in which the internal solution flow is independent of time, and incompressibility, which is the assumption that the solution density does not change to a constant. [ka]

[0090] The density of the mixed working fluid can be calculated by linear interpolation based on the concentration, but as mentioned above, the difference in viscosity coefficients during mixing cannot be predicted by linear interpolation due to the hydration layer that occurs when the water and oil are not mixed well. Therefore, the viscosity values were calculated using Lagrange interpolation using an Nth-order polynomial for the viscosity values based on the ethanol concentration in Table 1. The applied boundary conditions reflected the flow conditions used in the experiment and in the production of nanolipid particles. A non-slip condition, where there is no flow velocity, was applied to the channel wall in the mixer, and an open boundary condition, where the gauge pressure was 0 atm and 1 atm, was applied to the outlet, making it easier to calculate the flow field, i.e., the flow of the internal solution.

[0091] The solution flow rate (u) calculated from the analysis results was used in the mass continuity equation to calculate the mixing index according to the ethanol concentration distribution, reflecting the coupling between molecular diffusion at the microscopic molecular level and advection due to fluid flow, using the diffusion analysis law (Equation 3).

[0092] As can be seen from the results of Figure 8, it was confirmed that high-quality lipid nanoparticles containing active ingredients can be efficiently produced by utilizing the structure of the mixer unit of the chip for producing lipid nanoparticles according to one embodiment of the present invention. In particular, it was confirmed that sufficient mixing was possible even with just a mixing section of 30 to 40 mm in length.

[0093] Fig. 9 is a flowchart illustrating a method for producing lipid nanoparticles according to one embodiment of the present invention. Fig. 10 is a flowchart illustrating in detail the steps of producing lipid nanoparticles on a chip in the method for producing lipid nanoparticles of Fig. 9.

[0094] 9 and 10, the method for producing lipid nanoparticles may include a step S100 of preparing raw materials, a step S200 of producing lipid nanoparticles on a chip, and a post-treatment step S300.

[0095] In the step S100 of preparing raw materials, a first raw material containing mRNA and a second raw material containing lipid may be prepared.

[0096] In the step S200 of producing lipid nanoparticles on the chip, the first raw material and the second raw material may be mixed to form lipid nanoparticles containing mRNA.

[0097] In the post-treatment step S300, the lipid nanoparticle-containing solution may be subjected to diafiltration, further concentrated to a required concentration, and filled into individual containers to produce a final product.

[0098] Here, the step S200 of forming lipid nanoparticles may be performed on a chip for preparing lipid nanoparticles having a flow path formed therein, and may include an initial mixing step S210, a stabilizing step S220, and a mixing step S230. The chip for preparing lipid nanoparticles may be the chip for preparing lipid nanoparticles described with reference to FIG. 1, etc. For example, the chip for preparing lipid nanoparticles may include a mixer unit including a stabilizing unit and a mixing unit. The chip for preparing lipid nanoparticles may be the chip for preparing lipid nanoparticles described with reference to FIGS. 1 and 2.

[0099] In the initial mixing step S210, the first raw material and the second raw material may be initially mixed in a merging section of a mixer section of the chip for producing lipid nanoparticles.

[0100] In the stabilization step (S220), the fluid in the mixer may pass through the merging unit and then through a stabilizing unit. The stabilizing unit may include a left stabilizing channel and a right stabilizing channel having the same width and length, and the left stabilizing channel and the right stabilizing channel may be symmetrically oval or circular.

[0101] In the mixing step (S230), the fluid in the mixer unit may pass through the stabilizing unit and then the mixing unit. The stabilizing and mixing steps may be repeated several times depending on the design of the mixer unit. Through this process, the first raw material and the second raw material may be mixed, and lipid nanoparticles may be produced through a self-assembly process.

[0102] Although the present invention has been described with reference to the above embodiments, it will be understood by those skilled in the art that various modifications and variations can be made thereto without departing from the spirit and scope of the present invention as set forth in the following claims. The present application includes the following aspects. [Section 1] a first raw material supply channel; A second raw material supply channel, and a mixer unit connected to the first raw material supply passage and the second raw material supply passage, for mixing the first raw material supplied through the first raw material supply passage and the second raw material supplied through the second raw material supply passage; The mixer unit includes: a first stabilization section, and a first mixing section connected to the first stabilizing section, in which the first raw material and the second raw material are mixed with each other; A chip for producing lipid nanoparticles, characterized in that the mixing of the first raw material and the second raw material is performed more in the first mixing section than in the first stabilization section. [Section 2] Item 1, characterized in that the first stabilizing section includes a left stabilizing channel having a first width and a first length and a right stabilizing channel having the first width and the first length. A chip for producing lipid nanoparticles as described in item 1. [Section 3] Item 3. The chip for producing lipid nanoparticles according to item 2, wherein the left stabilizing channel and the right stabilizing channel of the first stabilizing section are symmetrically elliptical or circular. [Section 4] The first mixing section includes a left mixing channel having a second width and a right mixing channel having a third width, and the second width and the third width are different from each other. [Section 5] Item 5. The chip for producing lipid nanoparticles according to item 4, wherein the left and right mixing channels of the first mixing section are elliptical or circular. [Section 6] the first stabilization unit is connected to the first raw material supply flow path and the second raw material supply flow path, The first raw material flows through the first raw material supply flow path, the first stabilization section, and the first mixing section in that order, and the second raw material flows through the second raw material supply flow path, the first stabilization section, and the first mixing section in that order. The first raw material and the second raw material are mixed at the interface between the first raw material and the first raw material, but the mixing of the first raw material and the second raw material is more frequent in the first mixing section than in the first stabilization section. [Section 7] the first mixing section is connected to the first raw material supply flow path and the second raw material supply flow path, Item 1, characterized in that the first raw material flows through the first raw material supply flow path, the first mixing section, and the first stabilization section in that order, and the second raw material flows through the second raw material supply flow path, the first mixing section, and the first stabilization section in that order. [Section 8] The mixer unit includes: a second stabilization section coupled to the first mixing section; and Item 1. The chip for producing lipid nanoparticles according to item 1, further comprising a second mixing section connected to the second stabilizing section. [Section 9] a first raw material supply unit that supplies a first raw material; a second raw material supply unit that supplies a second raw material; a chip for producing lipid nanoparticles, the chip including a mixer unit that mixes the first raw material and the second raw material to form a mixed liquid; and A lipid nanoparticle obtaining unit for obtaining lipid nanoparticles produced from the lipid nanoparticle producing chip, The mixer unit of the chip for producing lipid nanoparticles comprises a first stabilizing unit, and A lipid nanoparticle manufacturing system, characterized in that it includes a first mixing section connected to the first stabilization section, in which the first raw material and the second raw material are mixed with each other. [Section 10] Item 10. The lipid nanoparticle producing system described in Item 9, characterized in that the first stabilizing section includes a left stabilizing channel having a first width and a first length and a right stabilizing channel having the first width and the first length, and the first mixing section includes a left mixing channel having a second width and a right mixing channel having a third width, and the second width and the third width are different from each other. [Section 11] Preparing a first ingredient containing an active ingredient and a second ingredient containing a lipid; mixing the first ingredient with the second ingredient to form lipid nanoparticles containing the active ingredient; and A post-processing step of filtering the solution containing the lipid nanoparticles and filling it into individual containers to produce a final product, The step of forming the lipid nanoparticles is performed on a chip for producing lipid nanoparticles, in which a channel is formed; a stabilization step in which the first and second feedstocks pass through a stabilization section including a left stabilization channel and a right stabilization channel having the same width and length; and A method for producing lipid nanoparticles, comprising a mixing step in which the first raw material and the second raw material are mixed with each other by passing through a mixing section including a left mixing channel and a right mixing channel having different widths. [Section 12] The left stabilizing channel and the right stabilizing channel of the stabilizing section are symmetrical to each other and have an elliptical or circular shape, Item 12. The method for producing lipid nanoparticles according to Item 11, wherein the left mixing channel and the right mixing channel of the mixing section are elliptical or circular. [Section 13] Item 12. The method for producing lipid nanoparticles according to Item 11, characterized in that the stabilization step and the mixing step are alternately repeated at least twice or more. [Explanation of symbols]

[0103] 10: First raw material supply section 20: Second raw material supply section 30: Lipid nanoparticle acquisition section 100: Lipid nanoparticle manufacturing chip 200: Mixer section 210: First stabilization section 220: First mixing section 230: Second stabilization section 240: Second mixing section

Claims

1. A first raw material supply flow path containing an active ingredient; a second feedstock supply channel containing a lipid; and a mixer unit connected to the first raw material supply passage and the second raw material supply passage, for mixing the first raw material supplied through the first raw material supply passage and the second raw material supplied through the second raw material supply passage; The mixer unit includes: a first stabilization section including a left stabilization channel having a first width and a first length and a right stabilization channel having the first width and the first length, in which lipid nanoparticles are formed and stabilized by a self-assembly process; and a first mixing section connected to the first stabilizing section, the first mixing section including a left mixing channel having a second width and a right mixing channel having a third width, the second width and the third width being different from each other, in which the first raw material and the second raw material are mixed with each other by a Dean vortex; A chip for producing lipid nanoparticles, characterized in that the mixing of the first raw material and the second raw material is performed more in the first mixing section than in the first stabilization section.

2. The chip for producing lipid nanoparticles according to claim 1 , wherein the left stabilizing channel and the right stabilizing channel of the first stabilizing section are symmetrically elliptical or circular.

3. The chip for producing lipid nanoparticles according to claim 1 , wherein the left and right mixing channels of the first mixing unit are elliptical or circular.

4. the first stabilization unit is connected to the first raw material supply flow path and the second raw material supply flow path, The first raw material flows through the first raw material supply flow path, the first stabilization section, and the first mixing section in that order, and the second raw material flows through the second raw material supply flow path, the first stabilization section, and the first mixing section in that order. The first raw material and the second raw material are mixed at the interface between the first raw material and the second raw material, but the mixing of the first raw material and the second raw material is more intense in the first mixing section than in the first stabilization section. The lipid nanoparticle production chip according to claim 1, characterized in that:

5. the first mixing section is connected to the first raw material supply flow path and the second raw material supply flow path, The first raw material flows through the first raw material supply flow path, the first mixing section, and the first stabilization section in that order, and the second raw material flows through the second raw material supply flow path, the first mixing section, and the first stabilization section in that order. The chip for producing lipid nanoparticles according to claim 1.

6. The mixer unit includes: a second stabilization section coupled to the first mixing section; and The chip for producing lipid nanoparticles according to claim 1 , further comprising a second mixing section connected to the second stabilizing section.

7. A first raw material supply unit that supplies a first raw material containing an active ingredient; a second raw material supply unit that supplies a second raw material containing lipid; a chip for producing lipid nanoparticles, the chip including a mixer unit that mixes the first raw material and the second raw material to form a mixed liquid; and A lipid nanoparticle obtaining unit for obtaining lipid nanoparticles produced from the lipid nanoparticle producing chip, The mixer unit of the chip for producing lipid nanoparticles includes a left stabilizing channel having a first width and a first length and a right stabilizing channel having the first width and the first length, and a first stabilizing unit in which lipid nanoparticles are formed and stabilized by a self-assembly process; and a first mixing section connected to the first stabilizing section, the first mixing section including a left mixing channel having a second width and a right mixing channel having a third width, the second width and the third width being different from each other, in which the first raw material and the second raw material are mixed with each other by a Dean vortex; A lipid nanoparticle manufacturing system, characterized in that the mixing of the first raw material and the second raw material is performed more in the first mixing section than in the first stabilization section.

8. providing a first ingredient containing an active ingredient and a second ingredient containing a lipid; mixing the first ingredient with the second ingredient to form lipid nanoparticles containing the active ingredient; and A post-processing step of filtering the solution containing the lipid nanoparticles and filling it into individual containers to produce a final product, The step of forming the lipid nanoparticles is performed on a chip for preparing lipid nanoparticles, in which a channel is formed; a stabilization step in which the first raw material and the second raw material pass through a stabilization section including a left stabilization channel and a right stabilization channel having the same width and length, and lipid nanoparticles are formed and stabilized through a self-assembly process; and a mixing step in which the first raw material and the second raw material pass through a mixing section including a left mixing channel and a right mixing channel having different widths and are mixed with each other by a Dean vortex; A method for producing lipid nanoparticles, characterized in that the mixing of the first raw material and the second raw material is performed more in the mixing section than in the stabilization section.

9. The left stabilizing channel and the right stabilizing channel of the stabilizing section are symmetrical to each other and have an elliptical or circular shape, The method for producing lipid nanoparticles according to claim 8, wherein the left and right mixing channels of the mixing section are elliptical or circular.

10. The method for producing lipid nanoparticles according to claim 8, wherein the stabilizing step and the mixing step are alternately repeated at least two times.

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