Apparatus and method for manufacturing composite

Abstract

The present disclosure provides an apparatus and a method for manufacturing a composite. In one aspect, the present disclosure provides a method for manufacturing a formulation comprising a first component and a second component. This method includes: a step for preparing a series of flow paths in which a first flow path through which a first fluid flows at a first flow velocity and a second flow path through which a second fluid flows at a second flow velocity are combined at a merging portion to form a third flow path through which a third fluid flows at a third flow velocity; a step in which the first component is injected into the first flow path at a first time; and a step in which the second component is injected into the second flow path at a second time. The first time and the second time are set so that the first component and the second component reach the merging portion at a timing when the components are mixed.

Description

Apparatus and method for manufacturing composites

[0001] The present disclosure relates to an apparatus and method for manufacturing a composite, and more particularly to a technique for manufacturing a composite by mixing fluids flowed through at least two channels.

[0002] As a technique for producing sterile liposomes, "in-line liposome production technology," which continuously forms liposomes or lipid nanoparticles (LNPs) in a closed capillary, has attracted attention, and a technique for adjusting the concentration of formed liposomes in-line has been developed (Reference 1). However, in order to obtain lipid particles with suitable particle size and other factors, various parameters such as flow rate must be adjusted, and the parameters set for small-scale sample preparation at the laboratory level often differ from the parameters suitable for mass production for product supply.

[0003] Therefore, conventionally, manufacturing conditions for supplying products have had to be adjusted separately, consuming a large amount of materials.

[0004] International Publication No. 2016 / 024510

[0005] The present inventors have conducted extensive research into a method for reflecting the conditions for preparing a small amount of lipid particle samples in the manufacturing conditions for supplying the product, in order to reduce the need for additional studies to determine the manufacturing conditions for supplying the product, and have arrived at the device and method of the present disclosure. Therefore, the present disclosure provides a device and method for producing drug-loaded lipid particles by mixing fluids flowed from at least two flow paths.

[0006] Accordingly, the present disclosure provides the following: (Item 1) A method for producing a formulation containing a first component and a second component, comprising: preparing a series of flow paths, where a first flow path for flowing a first fluid at a first flow rate and a second flow path for flowing a second fluid at a second flow rate are joined at a junction to form a third flow path for flowing a third fluid at a third flow rate; injecting a sample containing the first component into the first flow path at a first time; and injecting a sample containing the second component into the second flow path at a second time, wherein the first and second times are set so that the first and second components reach the junction just as they are mixed. (Item 2) The method of any of the above items, wherein the first component is injected into a middle portion of the first flow path and the second component is injected into a middle portion of the second flow path. (Item 3) The method of any of the above items, wherein the third time is set based on the first and second times. (Item 4) Any of the methods above, wherein the first flow rate or the second flow rate exceeds approximately 2.4 mL / min. (Item 5) Any of the methods above, wherein the series of flow paths includes a fourth flow path through which a sample containing a third component flows, and the method further includes the step of injecting the third component into the fourth flow path at a third time, wherein the first time, the second time, and the third time are set so that the first component, the second component, and the third component reach the junction at a timing at which they are mixed. (Item 6) Any of the methods above, wherein the third flow path and the fourth flow path join at an additional junction downstream of the junction. (Item 7) Any of the methods above, wherein the first flow path, the second flow path, and the fourth flow path join at the junction. (Item 8) Any of the methods above, further including the step of detecting a junction state of the first component and the second component downstream of the junction. (Item 9) The method of any of the above items, wherein the confluence state is detected as a state selected from the group consisting of the concentration of at least one of the first component and the second component, the particle diameter of particles in the flow, and the state of chemical bonding between the first component and the second component.(Item 10) Any of the methods described above, wherein in the step of injecting a sample containing the first component into the first flow path, the first flow path is in a liquid-flowing state, and in the step of injecting a sample containing the second component into the second flow path, the second flow path is in a liquid-flowing state. (Item 11) Any of the methods described above, wherein in the step of injecting a sample containing the first component into the first flow path and the step of injecting a sample containing the second component into the second flow path, the retention times from when the sample containing the first component and the sample containing the second component are filled in the sample injection section until they are injected into the flow paths are independently within one minute. (Item 12) Any of the methods described above, wherein the times from when the first component and the second component are injected until they reach the confluence are independently within two seconds. (Item 13) Any of the methods described above, wherein the step of injecting the first component into the first flow path injects 1000 μg or less of the first component, and the step of injecting the second component into the second flow path injects 1000 μg or less of the second component. (Item 14) Any of the methods described above, wherein a fourth fluid flowing through a fifth flow path is joined with the third fluid at an additional junction to form a sixth flow path through which the fifth fluid flows. (Item 15) Any of the methods described above, wherein the junction includes a T-mixer, a microfluidic device, or a static mixer. (Item 16) Any of the methods described above, wherein the third flow path is equipped with a mixer. (Item 17) Any of the methods described above, wherein the mixer on the third flow path is a static mixer. (Item 18) Any of the methods above, wherein the first flow path and the second flow path each include a plurality of divided flow paths, and the confluence section includes a plurality of fractional flow sections where the divided flow paths of the first flow path and the divided flow paths of the second flow path join together. (Item 19) Any of the methods above, wherein the divided flow paths are formed by branching the first flow path and / or the second flow path. (Item 20) Any of the methods above, wherein in the third flow path, divided flow paths downstream of the fractional flow sections join together. (Item 21) Any of the methods above, further comprising the step of withdrawing a fraction containing a compound of the first component and the second component from a collection section of the third flow path at a third time.(Item 22) The method of any of the above items, wherein the collection portion is positioned in the middle of the third flow path. (Item 23) The method of any of the above items, further comprising the step of distributing the fractions to a receiver. (Item 24) The method of any of the above items, wherein the distributing step comprises distributing a plurality of the fractions to the receiver comprising a plurality of compartments. (Item 25) The method of any of the above items, further comprising the step of inspecting a plurality of the fractions. (Item 26) The method of any of the above items, wherein the time at which the fractions are removed from the collection portion is controlled based on the time and flow rate at which components are injected into the flow path. (Item 27) An apparatus for injecting multiple samples into multiple flow paths at a timing when a first component and a second component are mixed, comprising: a first flow path for flowing a first fluid, a first injection unit for injecting a sample containing the first component into the first flow path, a second flow path for flowing a second fluid, a second injection unit for injecting a sample containing the second component into the second flow path, a junction where the first flow path and the second flow path are joined, a third flow path extending from the junction for flowing a third fluid, and a control unit, wherein the control unit is configured to control the time when the first injection unit injects the first component into the first flow path and the time when the second injection unit injects the second component into the second flow path. (Item 28) The apparatus of any of the above items, wherein the first injection unit is positioned in a central portion of the first flow path, and the second injection unit is positioned in a central portion of the second flow path. (Item 29) The device of any of the above items, wherein the first injection unit is a valve capable of injecting a first component into the first flow path within 5 seconds, and the second injection unit is a valve capable of injecting a second component into the second flow path within 5 seconds. (Item 30) The device of any of the above items, wherein at least one of the first injection unit and the second injection unit can switch flow paths within about 500 ms.(Item 31) The device according to item 19, further comprising: a fourth flow path for flowing a third fluid; and a third injection unit for injecting a sample containing a third component into the fourth flow path, wherein the series of flow paths includes the fourth flow path, and the control unit is configured to control the time for the first injection unit to inject the first component into the first flow path, the time for the second injection unit to inject the second component into the second flow path, and the time for the third injection unit to inject the third component into the fourth flow path. (Item 32) Any of the devices above, including a detector downstream of the junction. (Item 33) Any of the devices above, wherein the first injection unit includes a valve with a sample loop having a capacity of 5 mL or less, and the second injection unit includes a valve with a sample loop having a capacity of 5 mL or less. (Item 34) Any of the devices above, wherein the pump for flowing the first fluid and the second fluid is a plunger pump. (Item 35) The device of any of the above items, wherein the back pressure applied to the pumps for flowing the first fluid and the second fluid is 1 MPa or more. (Item 36) The device of any of the above items, further comprising: a fourth flow path for flowing a fourth fluid; an additional junction where the fourth flow path and the third flow path join; and a fifth flow path extending from the additional junction for flowing a fifth fluid. (Item 37) The device of any of the above items, wherein the third flow path comprises a collection section, and the collection section further comprises a collection device. (Item 38) The device of any of the above items, further comprising a constant temperature bath that maintains the first, second, and third flow paths, the first and second injection sections, and at least a portion of the junction at a constant temperature. (Item 39) The device of any of the above items, wherein the control unit further controls the collection time of the collection device. (Item 40) The device of any of the above items, wherein the control unit controls at least one of the collection start time and the collection time interval of the collection device. (Item 41) The device of any of the above items, wherein the collection unit is positioned in the middle of the third flow path. (Item 42) The device of any of the above items, wherein the collection device includes a valve. (Item 43) The device of any of the above items, wherein the valve is a sliding solenoid valve.(Item 44) The device of any of the above items, further comprising a receiver for receiving the fractions removed from the collection device. (Item 45) The device of any of the above items, wherein the receiver comprises a plurality of compartments for receiving a plurality of fractions. (Item 46) A program for injecting multiple components into multiple flow paths connected to a confluence, the program being executed in an apparatus having a processor, the program causing the processor to perform processes including: receiving information about a first flow path and information about a second flow path; determining a first time for injecting a sample containing a first component into the first flow path and a second time for injecting a sample containing a second component into the second flow path based on the information about the first flow path and the information about the second flow path, wherein the first time and the second time are times such that the first component injected into the first flow path and the second component injected into the second flow path reach the confluence at a timing where they are mixed; instructing an injection device to inject the sample containing the first component into the first flow path at the first time; and instructing the injection device to inject the sample containing the second component into the second flow path at the second time. (Item 47) The program of any of the above items, wherein the processing further includes: receiving information about a third flow path downstream of the junction; determining a third time for removing a fraction from the third flow path based on the information about the third flow path; and instructing a collection device to remove the fraction from the third flow path at the third time.(Item 48) A recording medium storing a program for causing an apparatus having a processor to execute a method for injecting multiple components into multiple flow paths connected to a confluence, the method comprising: receiving information about a first flow path and information about a second flow path; determining a first time for injecting a sample containing a first component into the first flow path and a second time for injecting a sample containing a second component into the second flow path based on the information about the first flow path and the information about the second flow path, wherein the first time and the second time are times such that the first component injected into the first flow path and the second component injected into the second flow path reach the confluence at a timing where they are mixed; instructing an injection device to inject the sample containing the first component into the first flow path at the first time; and instructing the injection device to inject the sample containing the second component into the second flow path at the second time. (Item 49) The recording medium of any of the above items, wherein the method further includes: receiving information about a third flow path downstream of the junction; determining a third time for withdrawing a fraction from the third flow path based on the information about the third flow path; and instructing a collection device to withdraw the fraction from the third flow path at the third time. (Item 50) A method for screening manufacturing conditions for lipid particles loaded with drugs, comprising: preparing a series of flow paths, in which a first flow path for flowing a first fluid at a first flow rate and a second flow path for flowing a second fluid at a second flow rate join at a confluence to form a third flow path for flowing a third fluid at a third flow rate; (1) injecting a sample containing a candidate lipid into the first flow path at a first time; (2) injecting a sample containing a candidate drug into the second flow path at a second time; (3) extracting a fraction containing candidate lipid particles from a collection portion of the third flow path at a third time; and evaluating the candidate lipid particles, wherein the first time and the second time are set so that the candidate lipid and the candidate drug arrive at the confluence simultaneously.(Item 51) Any of the methods above, further comprising the steps of preparing a plurality of candidate lipid particles by repeating steps (1) to (3), comparing the plurality of candidate lipid particles, and selecting a hit lipid particle. (Item 52) A method for producing any of the hit lipid particles above under production conditions set in the screening method of any of the above. (Item 53) A method for producing any of the hit lipid particles above, wherein the production amount of the hit lipid particle is increased by increasing the frequency of performing steps (1) to (3).

[0007] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0008] According to the present disclosure, suitable reaction conditions (e.g., lipid particle formation conditions) can be determined with the consumption of small amounts of materials, and the production amount can be easily scaled up using those conditions, thereby reducing material consumption and flexibly responding to the needs of providing different products.

[0009] FIG. 1B is a diagram showing an example of an apparatus (system) of the present disclosure. 101: autosampler, 102: injection section, 103: pump, 104: flow path, 105: confluence section, 106: control section, 107: collection device, 108: valve, 109: receiver. FIG. 1C is a diagram showing a modified example of the apparatus (system) of FIG. 1A. 104c': divided flow path, 105': fractional flow section. FIG. 1D is a diagram showing an example of an apparatus (system) of the present disclosure. This embodiment includes a computer (110) and a detector (111) in addition to the configuration of FIG. 1. This is a block diagram showing the configuration of a lipid particle size control unit according to function in one embodiment of the present invention. This is a block diagram showing the configuration of a lipid particle size control unit according to function in one embodiment of the present invention. This shows a study to achieve appropriate mixing of two samples during simultaneous injection timing. Each graph shows the absorbance of nucleic acid and lipid in successive fractions obtained. FIG. 4A shows the results when tubing with the same inner diameter and length is attached to the two flow paths. Figure 4B shows the results when a tubing with an inner diameter of 0.381 mm and a length of 76 cm (87 μL) was added to the channel for injecting the nucleic acid sample. Figure 4C shows the results when the tubing added to the channel for injecting the nucleic acid sample was changed to one with an inner diameter of 0.381 mm and a length of 134 cm (153 μL). Figure 4D shows the results when a tubing with an inner diameter of 0.381 mm and a length of 76 cm (87 μL) was added to the channel for injecting the nucleic acid sample, and the tubing from the valve to the mixer in the channel for injecting the lipid sample was changed to a smaller volume tubing with an inner diameter of 0.18 mm and a length of 6 cm. This shows the results of achieving proper mixing of the two samples when tubing with the same inner diameter and length is used for each channel. Each graph shows the absorbance of nucleic acid and lipid in consecutive fractions obtained. Figure 5A shows the results when a sample injection signal is sent to two valves simultaneously. 5B shows the results when the injection timing of the nucleic acid sample was delayed by 100 msec from the injection timing of the lipid sample. The graph shows the change in nucleic acid and lipid concentrations in the collected fractions when the valve was changed. The vertical axis shows absorbance at a wavelength of 260 nm or 600 nm, and the horizontal axis shows the number of the collected fractions. The graph shows the change in particle size distribution of lipid particles in the collected fractions when the valve was changed.1 shows an outline of the device configuration of Example 6 in which three components are mixed using four pumps.

[0010] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to be used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) shall prevail. Below, definitions and / or basic technical content of terms particularly used in this specification will be explained as appropriate.

[0011] (Definitions, etc.) In this specification, the term "flow path" refers to a generally tubular structure through which a fluid flows. In this specification, a flow path typically refers to a series of structures from a mechanism that draws a fluid into a tube, such as a pump, to a confluence, from a confluence to another confluence, or from a mechanism that draws a fluid into a tube or from a confluence to an outlet where the fluid is released. For example, even if a mechanism that can temporarily cut off the flow, such as a valve or a collection section, is present along the way, it can be considered a single flow path. It does not necessarily have to be composed of a single member, and may include connecting members such as connecting tubes. Typically, the entire interior of a single flow path is fluid-communicating. Typically, a flow path does not have any parts where the fluid comes into contact with the outside other than the inlet and outlet.

[0012] In this specification, the term "junction" refers to a portion where fluids flow in from multiple flow paths, and is typically connected to one or more flow paths through which fluids flow out of the junction. At the junction, multiple fluids are usually mixed, and therefore the junction is sometimes referred to as a mixer.

[0013] As used herein, the term "flow rate" refers to the speed at which a solution flows through a tube, and is a quantity that can be expressed in either the dimension of (distance) / (time) or (volume) / (time).

[0014] As used herein, the term "retention time" refers to the time it takes for a solution to move from one region to another. For example, if region A is connected to region B via a liquid transfer tube C, the retention time from region A to region B can be calculated by dividing the length of the liquid transfer tube by the flow rate (cm / min).

[0015] As used herein, the term "complex" refers to an entity formed by the contact of multiple components, and examples thereof include drug-loaded lipid particles composed of lipids and drugs, products formed by chemical reactions between different compounds, and complexes formed between different proteins.

[0016] As used herein, the term "complex" or "complex molecule" refers to any construct comprising two or more moieties. For example, if one moiety is a polypeptide, the other moiety may be either a polypeptide or another substance (e.g., sugar, lipid, nucleic acid, other carbohydrate, etc.). As used herein, the two or more moieties constituting the complex may be bonded by a covalent bond or by other bonds (e.g., hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). When two or more moieties are polypeptides, they may also be referred to as chimeric polypeptides. Thus, as used herein, "complex" includes molecules formed by linking multiple types of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules.

[0017] As used herein, the term "lipid particle" refers to a lipid vesicle formed from a lipid bilayer containing lipid molecules. Specifically, it refers to a vesicle having a space separated from the outside world by a lipid bilayer formed based on the polarity of the hydrophobic and hydrophilic groups of the lipid molecules. The lipid particle may be a unilamellar liposome (SUV: Small Unilamellar Vesicle) having a single lipid bilayer membrane, a multilamellar liposome (MLV: Multilamellar Vesicle) having multiple layers, or a lipid nanoparticle in the form of a lipid-filled core coated with a lipid monolayer. The bilayer membrane is composed of two monolayer lipid membranes having a hydrophobic "tail" region and a hydrophilic "head" region. The membrane bilayer structure is such that the hydrophobic (nonpolar) "tail" of the lipid monolayer faces the center of the bilayer, while the hydrophilic "head" faces the aqueous phase. Lipid bilayer membranes are similar to the cell membranes that make up living organisms and are therefore easily absorbed into the in vivo environment. Therefore, liposomes are sometimes used in drug delivery systems (DDS). For example, drugs can be encapsulated in liposomes to transport the drugs to a specific site in the body. The liposomes of the present disclosure are particularly suitable for pharmaceutical applications, but their applications are not limited thereto and may also be used in food, cosmetics, agriculture, imaging, and the like.

[0018] As used herein, when a lipid particle is said to be "loaded" with a drug, the drug is held inside and / or on the surface of the lipid particle. When a lipid particle is "loaded" with a drug, at least a portion of the drug is held in the lipid bilayer membrane inside or on the surface of the lipid particle. The drug may be present in the aqueous phase within the lipid particle, or may be present in a state where it is immobilized on the surface of the lipid layer by electrostatic interactions or the like, or may be partly or entirely contained within the lipid layer.

[0019] As used herein, the term "lipid" is used in the usual sense in the art and refers to a substance having a hydrophobic moiety such as a long-chain fatty acid or a hydrocarbon chain. Examples of lipids include phosphatidylcholines (soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, etc.), phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, phosphasphingomyelins, phosphatidic acids, long-chain alkyl phosphates, gangliosides, glycolipids, phosphatidylglycerols, cholesterols (cholesterol, phytosterols (sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol), lanosterol, ergosterol, and their fatty acid esters), tocopherol, steroids, fatty acids, and fatty acid esters of glycerol. Lipids include amphipathic lipids (such as phospholipids, aminolipids, sphingolipids, glycosphingolipids, diacylglycerols, and β-acyloxyacids), anionic lipids (such as phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups attached to neutral lipids), cationic lipids (such as N,N-dioleyl-N,N-dimethylanthracene chloride), and hydroxypropyltrimonials (such as hydroxypropyltrimonials). ammonium chloride ("DODAC"), N-(2,3-oleyloxy)propyl-N,N,N-trimethylammonium chloride ("DOTMA"), N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"), N-(2,3-oleyloxy)propyl-N,N,N-trimethylammonium chloride ("DOTAP"), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), and N(1,2-dimyristyloxyprop-3-yl)N,N-dimethylhydroxyethylammonium bromide ("DMRIE")). Lipids also include the ionizable lipids D-Lin-MC3-DMA, ALC-0315, and SM-102.

[0020] As used herein, the terms "drug," "agent," and "factor" (all of which correspond to the English term "agent") are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, or electricity) that can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins (including antibodies, etc.), polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals, etc.), and composite molecules thereof.

[0021] Generally, the compositions, medicaments, agents (therapeutic agents, prophylactic agents, etc.) of the present disclosure comprise a therapeutically effective amount of a medicament or active ingredient, and a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0022] As used herein, the term "drug" is used in the ordinary sense used in the art, and refers to a substance that exerts some physiological effect when administered to an organism, and includes, for example, proteins (including enzymes, antibodies, etc.), peptides, nucleic acids (DNA, mRNA, siRNA, miRNA), vectors, viral particles, plasmids, toxins, saccharides (oligosaccharides and polysaccharides), polymeric compounds, anticancer drugs, antibiotics, enzymes, antioxidants, lipid uptake inhibitors, hormones, anti-inflammatory drugs, steroids, vasodilators, angiotensin converting agents, and the like. Reductase inhibitors, angiotensin receptor antagonists, smooth muscle cell proliferation and migration inhibitors, platelet aggregation inhibitors, anticoagulants, chemical mediator release inhibitors, vascular endothelial cell proliferation promoters or inhibitors, aldose reductase inhibitors, mesangial cell proliferation inhibitors, lipoxygenase inhibitors, immunosuppressants, immunostimulants, antirheumatic drugs, anti-inflammatory enzyme preparations, gout treatment drugs, antihistamines, chemical mediator release inhibitors, antiviral agents, Maillard reaction inhibitors, amyloidosis inhibitors, nitric oxide synthesis inhibitors, AGFs (Advanced glycation endproducts inhibitors, hemoglobin, radical scavengers, glycosaminoglycans and their derivatives, corticosteroids and their derivatives such as prednisolone, methylprednisolone, and dexamethasone, non-steroidal anti-inflammatory drugs such as aspirin, indomethacin, ibuprofen, mefenamic acid, and phenylbutazone, mesangial cell proliferation inhibitors such as heparin and low molecular weight heparin, immunosuppressants such as cyclosporine, ACE (angiotensin converting enzyme) inhibitors such as captopril, AGE (advanced glycation endoproduct) inhibitors such as methylguanidine, TGF-β antagonists such as biglycan and decorin, and PKC (protein kinase C) inhibitors. C) inhibitors, prostaglandin preparations such as PGE1 and PGI2, papaverine drugs, nicotinic acid drugs, tocopherol drugs, and peripheral vasodilators such as calcium channel blockers, phosphodiesterase inhibitors, antithrombotic drugs such as ticlopidine and aspirin, anticoagulants such as warfarin, heparin, and antithrombin agents, thrombolytic drugs such as urokinase, chemical mediator release inhibitors, antibiotics, antioxidants, enzymes, lipid uptake inhibitors,Examples include hormones, vitamin C, vitamin E, radical scavengers such as SOD, and antisense oligonucleotides that have the effect of inhibiting the proliferation of mesangial cells.

[0023] As used herein, "particle size" or "particle diameter," as used in the ordinary sense in the art, is a measure used to express the size of a particle, and is a convenient value equivalent to the diameter of a particle when the particle is assumed to be a perfect sphere. The particle size of liposomes may be measured by any method known in the art, including, for example, the freeze-fracture method using a transmission electron microscope (TEM) and a method utilizing dynamic light scattering such as a Malvern Zetasizer. As used herein, the term "average particle size" may refer to any of the number-average particle size, volume-average particle size, or Z-average particle size, but unless otherwise specified, refers to the Z-average particle size calculated from measured particle sizes. As used herein, the term "particle size distribution" is used in the ordinary sense in the art and refers to the spread of particle sizes. The polydispersity index (PDI) is used as a measure of particle size distribution.

[0024] As used herein, "measurement" is used in the usual sense in the field, and refers to measuring and determining the amount of a certain object. As used herein, "detection" is used in the usual sense in the field, and refers to testing and finding a substance, component, etc., "identification" refers to the act of searching for an object's classification within existing classifications related to that object, and when used in the field of chemistry, refers to determining the identity of the target substance as a chemical substance (e.g., determining its chemical structure), and "quantitation" refers to determining the amount of the target substance present.

[0025] As used herein, the term "means" refers to any implement that can achieve a certain purpose (e.g., detection).

[0026] In this specification, the term "program" is used in the ordinary sense of the term in the art, and is a systematic description of the processes to be performed by a computer, and is legally treated as a "thing." All computers operate according to programs. In modern computers, programs are represented as data and stored on recording media or storage devices.

[0027] In this specification, the term "recording medium" refers to a recording medium that stores a program for executing the present disclosure, and the recording medium may be any type of recording medium as long as it can store the program. For example, the recording medium may be an internally stored ROM, an HDD, a magnetic disk, or an external storage device such as a flash memory such as a USB memory, but is not limited to these.

[0028] In this specification, the term "system" refers to a configuration that executes the method or program disclosed herein, and originally means a system or organization for accomplishing a purpose, in which multiple elements are systematically configured and influence each other; in the computer field, it refers to the entire configuration, including hardware, software, an OS, and a network.

[0029] As used herein, the term "about," when used herein in connection with a quantitative measurement other than temperature, refers to the stated value plus or minus 10%, or in some cases plus or minus 20% of the stated value, and when used herein in connection with temperature, refers to the stated value plus or minus 5°C.

[0030] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0031] This disclosure includes descriptions of various aspects, such as methods, devices, and systems, and a description of one aspect applies equally to other aspects. For example, a description of a method (such as flow rate) may also be considered a description of operating conditions of an apparatus or a description of instructions coded in a program.

[0032] (Method) In one aspect, the present disclosure provides a method for producing a formulation containing a first component and a second component. The method includes the steps of: preparing a series of flow paths, including a first flow path through which a first fluid flows at a first flow rate and a second flow path through which a second fluid flows at a second flow rate, the first flow path joining at a confluence to form a third flow path through which a third fluid flows at a third flow rate; injecting a sample containing the first component into the first flow path at a first time; and injecting a sample containing the second component into the second flow path at a second time. The first and second times are set so that the first and second components reach the confluence just as they are mixed. In one embodiment, the method further includes injecting a sample containing a third component into a fourth flow path at a third time, where the first, second, and third times may be set to arrive at a junction at a time when the first, second, and third components are mixed, and more components may be mixed in a similar manner. In one embodiment, the fourth flow path through which the third component flows may merge with the third flow path at an additional junction, where the third time for injecting the sample containing the third component into the fourth flow path is set to arrive at the junction at a time when a composite of the first and second components and the third component are mixed. The operation and characteristics of this fourth flow path are understood to be similar to those of the first and second flow paths.

[0033] Injecting a sample while fluid is flowing through a flow channel allows components to mix and form a composite under conditions that mimic mass production, even though only a small amount of components is consumed. Because mass production conditions typically involve high flow rates, the timing of sample injection must be controlled to ensure mixing of the components. As used herein, the "timing of mixing" or similar expressions describing the time during which different components flowing through different flow channels are mixed refers to the existence and / or range of time during which at least a portion of the components injected at a time come into contact with each other. Sample injection timings, such as the first and second times (typically referring to the time at which an injection unit such as a valve begins to operate), can be determined based on the timing of mixing. The injection timings of each sample, such as the first time, can vary depending on factors such as the set flow rate and the flow channel (inner diameter and length). Depending on the settings of these factors, the first and second times may ultimately be the same in some embodiments. The timing of injection is described in detail below.

[0034] When a sample is injected into a channel, the sample may have a certain volume, and the components in the sample move so that they are present along a certain length of the channel. Even if the components are uniformly dispersed in the sample before injection, injecting the sample into the channel causes the sample to come into contact with the fluid flowing through the channel from both the front and back. The components diffuse at the interface between the sample and the fluid (if the sample and the fluid are miscible, no clear interface is formed), resulting in non-uniform component concentrations in the channel, typically forming a mountain-shaped concentration distribution along the length of the channel. Typically, the first and second times can be selected so that the time spans during which fractions of fluid having concentrations 50% or more of the component concentrations in the sample containing the first and second components, respectively, pass through the inlet of the confluence at least partially overlap (e.g., so that at least about 50, about 60, about 70, about 80, about 90, or about 100% of the shorter time span overlaps with the longer time span). The first and second times can be determined based on the flow rates of the first and second flow paths and the lengths from the injection section for the sample containing the first component and the injection section for the sample containing the second component to the confluence. In one embodiment, the first and second times can be determined based on the flow rates of the first and second flow paths (V1 and V2), the injection volumes of the sample containing the first component and the sample containing the second component (W1S and W2S), and the internal volumes of the flow paths from each injection section to the confluence (W1L and W2L). In one embodiment, the flow times from the start time (T10 and T20) of the first and second components flowing into the confluence to the end time (T1E and T2E) of the flow (first component flow time T1: T1E to T10 and first component flow time T2: T2E to T20) are set so that the longer flow time is contained within the shorter flow time. For example, the following conditions are used:

[0035]

[0036] In this embodiment, when producing lipid nanoparticles (LNPs) loaded with nucleic acids, the amount of rare components (such as nucleic acids) used can be minimized by shortening the flow-through time. The flow-through time can be calculated, for example, by the following formula:

[0037]

[0038] The contact surface area between the sample and the fluid flowing through the channel during sample injection is preferably small (especially under the high flow rates encountered in mass production). For example, when injecting a sample by switching a valve, it is preferable that the time required for the valve to switch the channel is short, and can be completed within, for example, about 1000 ms, about 900 ms, about 800 ms, about 700 ms, about 600 ms, about 500 ms, about 400 ms, about 300 ms, about 200 ms, about 150 ms, about 100 ms, about 70 ms, or about 50 ms. Since sample injection requires a short amount of time, unless otherwise specified, the injection time in this specification refers to the time at which the sample begins to come into contact with the fluid flowing through the channel. The timing of sample injection may be automatically controlled by, for example, a PLC (Programmable Logic Controller) or manually controlled.

[0039] The disclosed method allows for the formation of a composite by injecting a sample, allowing for the investigation of various conditions simply by changing the type and / or concentration of components in the injected sample. For example, multiple samples can be prepared in advance, and composites prepared under different conditions can be easily obtained by sequentially injecting the samples using an autosampler and transferring them to the injection section of the flow channel. In one embodiment, a sample containing a first component is injected into the middle of a first flow channel (a portion of the flow channel that is neither the inlet nor the outlet), and / or a sample containing a second component is injected into the middle of a second flow channel.

[0040] In one embodiment, the first flow rate and / or the second flow rate are optimized for mass production and are fast relative to the amount of sample to be injected. Typically, the sample is injected into the flow channel (flow channel in a flowing state) without stopping the flow of the first and / or second fluid. Because fluid flow, such as shear force, can affect the properties (particle size, etc.) of the composite formed, it is preferable to be able to inject the sample without changing the flow rate as much as possible to enable scale-up under the same conditions. A short valve switching time is also preferable in this respect. In one embodiment, the first flow rate and / or the second flow rate are each independently about 1 mL / min, about 1.5 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 9 mL / min, about 10 mL / min, about 15 mL / min, about 20 mL / min, about 30 mL / min, about 40 mL / min, about 50 mL / min, about 60 mL / min, about 70 mL / min, about 8 The flow rate may be selected from 0 mL / min, about 90 mL / min, about 100 mL / min, about 200 mL / min, about 300 mL / min, about 400 mL / min, about 500 mL / min, about 600 mL / min, about 700 mL / min, about 800 mL / min, about 900 mL / min, about 1000 mL / min, about 2000 mL / min, about 3000 mL / min, about 4000 mL / min, about 5000 mL / min, or a range between any two of these values.

[0041] In one embodiment, the amount of sample injected at one time for the first flow path and / or the second flow path is an amount that, at the first flow rate and / or the second flow rate, independently, completes flow in about 50 ms, about 100 ms, about 150 ms, about 200 ms, about 300 ms, about 400 ms, about 500 ms, about 600 ms, about 700 ms, about 800 ms, about 900 ms, about 1000 ms, or a time (retention time) in the range between any two of these values. In one embodiment, the sample volume injected at one time for the first flow path and / or the second flow path can be independently selected from about 50 μL, about 100 μL, about 150 μL, about 200 μL, about 300 μL, about 400 μL, about 500 μL, about 600 μL, about 700 μL, about 800 μL, about 900 μL, about 1000 μL, about 2000 μL, about 5000 μL, about 10,000 μL, about 20,000 μL, about 50,000 μL, or a range between any two of these values. In one embodiment, the amount of component injected at one time for the first flow path and / or the second flow path can be independently selected from about 50 μg, about 100 μg, about 150 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1000 μg, about 2000 μg, about 5000 μg, about 10,000 μg, about 25,000 μg, or a range between any two of these values.

[0042] In one embodiment, in the step of injecting a sample containing a component (e.g., a first component) into a flow channel, the retention time from when the component is loaded into the sample injection section to when it is injected into the flow channel is independently about 0.5 minutes or less, about 1 minute or less, about 1.5 minutes or less, about 2 minutes or less, about 3 minutes or less, about 4 minutes or less, about 5 minutes or less, about 7 minutes or less, or about 10 minutes or less. In one embodiment, the time from when the sample containing the component (e.g., a first component) is injected into the flow channel (injection start time) to when it reaches the junction is independently about 0.1 seconds or less, about 0.2 seconds or less, about 0.5 seconds or less, about 1 second or less, about 1.5 seconds or less, about 2 seconds or less, about 3 seconds or less, about 4 seconds or less, about 5 seconds or less, about 7 seconds or less, or about 10 seconds or less.

[0043] In one embodiment, the fourth fluid flowing through the fifth channel may be joined with the third fluid at an additional joining point to form a sixth channel through which the fifth fluid flows. The addition of the fourth fluid may enable stabilization of the complex, etc. Additional fluids may be joined in a similar manner. The flow rates in the third and sixth channels can usually be automatically determined from the inner diameters of the first to sixth channels and the flow rates of the first, second, and fourth channels.

[0044] The types of conditions (e.g., flow rate, temperature, the type and concentration of components in the sample) considered for forming the complex in the method of the present disclosure can be appropriately set by those skilled in the art, and the appropriate values ​​of the parameters can be determined by the method of the present disclosure.For example, as shown in Figure 1, when producing lipid particles loaded with a drug, a sample containing a lipid (first component) in alcohol (such as ethanol) is injected into a first flow path through which alcohol (first fluid) flows, and a sample containing a drug (second component) such as nucleic acid in water is injected into a second flow path through which water (second fluid) flows, and a primary dilution is performed at the confluence of the first and second flow paths, and the primary dilution solution is passed through a third flow path for a predetermined time, and reaches the confluence of the third flow path and the fifth flow path through which water flows, and a secondary dilution is performed, and the complex can be collected from the sixth flow path through which the secondary dilution solution flows. In this case, the particle size of the lipid particles (e.g., average particle size: 10 to 1000 nm, 10 nm to 500 nm, 20 nm to 300 nm, 20 nm to 200 nm) can be controlled by the alcohol concentration in the primary diluent, the lipid concentration in the sample, the drug concentration in the sample, the predetermined time, the Reynolds number (Nre) in the third flow path, the pressure, the temperature during mixing, etc.

[0045] In one embodiment, the alcohol concentration in the primary diluent is preferably in a range that does not sufficiently stabilize the liposome membrane, while the alcohol concentration in the secondary diluent is preferably in a range that stabilizes the liposome membrane. In one embodiment, the alcohol concentration in the primary diluent may be 10 to 50 wt%, for example, about 10 wt%, about 15 wt%, about 18 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, or about 50 wt%. In one embodiment, the alcohol concentration in the secondary diluent may be 0 to 30 wt%, for example, about 0 wt%, about 5 wt% or less, about 10 wt% or less, about 15 wt% or less, about 18 wt% or less, about 20 wt% or less, about 25 wt% or less, or about 30 wt% or less. In one embodiment, the predetermined time for the primary diluent to pass through the third flow path (which may be controlled by at least one of the length and flow rate of the third flow path) may be from about 0.1 seconds to 60 minutes, e.g., about 0.1 seconds, about 0.2 seconds, about 0.5 seconds, about 1 second, about 2 seconds, about 5 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or a range between any two of these values, but may be longer. In one embodiment, the Reynolds number (N) in the third flow path may be less than 2000, less than 1000, less than 500, less than 300, less than 200, less than 100, or less than 50. In one embodiment, the pressure in the third flow path can be 0.5 MPa or more, 0.8 MPa or more, 1 MPa or more, 1.5 MPa or more, 2 MPa or more, 3 MPa or more, 4 MPa or more, 5 MPa or more, 8 MPa or more, or 10 MPa or more. In one embodiment, the surface of the lipid particles can be modified with a modifying agent. Each sample can also contain additives such as an osmotic pressure adjuster, a stabilizer, an antioxidant, or a pH adjuster.

[0046] In one embodiment, the method may further include detecting the confluence state of the first and second components downstream of the confluence (e.g., in the third flow path or the fifth flow path). The confluence state may be a state selected from the group consisting of the concentration of at least one of the first and second components, the particle size of particles in the flow, and the state of chemical bonding between the first and second components. Detection may be performed using a photodiode array (PDA) detector, an ultraviolet (UV) detector, a refractive index (RI) detector, a fluorescence detector, a circular dichroism detector, an optical rotation detector, or the like.

[0047] In one embodiment, the method may further include removing a fraction containing a composite of the first component and the second component from the collection portion of the third or sixth flow path at a third time. In one embodiment, the collection portion is located in the middle of the third or sixth flow path. In one embodiment, the method may further include distributing the fraction removed from the collection portion to a receiver. In one embodiment, the distributing step includes distributing multiple fractions to a receiver containing multiple compartments. In one embodiment, the method may include testing the multiple fractions. In one embodiment, the time at which the fraction is removed from the collection portion is controlled based on the time at which the components are injected into the flow path.

[0048] In one embodiment, the present disclosure provides a method for screening conditions for producing drug-loaded lipid particles. This method may include the steps of: preparing a series of flow paths, including a first flow path through which a first fluid flows at a first flow rate and a second flow path through which a second fluid flows at a second flow rate, and joining the first flow path at a junction to form a third flow path through which a third fluid flows at a third flow rate; (1) injecting a sample containing a candidate lipid into the first flow path at a first time; (2) injecting a sample containing a candidate drug into the second flow path at a second time; (3) withdrawing a fraction containing the candidate lipid particles from a collection section of the third flow path at a third time; and evaluating the candidate lipid particles. The first and second times may be set so that the candidate lipid and the candidate drug arrive at the junction simultaneously. In one embodiment, the method further includes the steps of preparing a plurality of candidate lipid particles by repeating steps (1) to (3), comparing the plurality of candidate lipid particles, and selecting a hit lipid particle. In one embodiment, the present specification also provides a method for producing hit lipid particles under the conditions of the flow path and flow rate set in the screening method. In one embodiment, the production amount of hit lipid particles can be increased by increasing the frequency of performing steps (1) to (3).

[0049] (Device / System) In one aspect, the present disclosure provides a device or system for injecting multiple samples into multiple flow channels at a timing at which a first component and a second component are mixed, and can be used in the methods described herein. The device or system may include a first flow channel for flowing a first fluid, a first injection unit for injecting a sample containing the first component into the first flow channel, a second flow channel for flowing a second fluid, a second injection unit for injecting a sample containing the second component into the second flow channel, a junction where the first and second flow channels join, a third flow channel extending from the junction for flowing a third fluid, and a controller. The controller may be configured to control the time at which the first injection unit injects the first component into the first flow channel and the time at which the second injection unit injects the second component into the second flow channel. In one embodiment, the device or system includes a fourth flow path for flowing a third component and a third injector for injecting the third component into the fourth flow path, and the controller may be configured to control the time for which the first injector injects the first component into the first flow path, the time for which the second injector injects the second component into the second flow path, and the time for which the third injector injects the third component into the fourth flow path, and similar injectors may be provided for injecting additional components. The operation and characteristics of this fourth flow path and injector are understood to be similar to those of the first and second flow paths and injectors.

[0050] In one embodiment, the first inlet is located in a middle of the first flow path, and the second inlet is located in a middle of the second flow path. In one embodiment, the device or system further includes a fifth flow path for flowing a fourth fluid, an additional junction where the fifth flow path and the third flow path join, and a sixth flow path for flowing the fifth fluid extending from the additional junction.

[0051] In the device or system of the present disclosure, the first and / or second injection section may include a valve. In one embodiment, the valves may each independently be a valve equipped with a sample injection pipe (such as a sample room) having a volume of about 0.05 mL or less, about 0.1 mL or less, about 0.15 mL or less, about 0.2 mL or less, about 0.3 mL or less, about 0.4 mL or less, about 0.5 mL or less, about 0.6 mL or less, about 0.7 mL or less, about 0.8 mL or less, about 0.9 mL or less, about 1 mL or less, about 2 mL or less, about 3 mL or less, about 4 mL or less, about 5 mL or less, about 7 mL or less, about 10 mL or less, about 20 mL or less, about 30 mL or less, about 40 mL or less, about 50 mL or less, about 100 mL or less, about 200 mL or less, about 300 mL or less, about 400 mL or less, or about 500 mL or less. In one embodiment, the valves are capable of injecting the first and / or second components into the first and / or second flow paths, independently, within about 0.05 seconds, about 0.1 seconds, about 0.15 seconds, about 0.2 seconds, about 0.3 seconds, about 0.4 seconds, about 0.5 seconds, about 0.6 seconds, about 0.7 seconds, about 0.8 seconds, about 0.9 seconds, or about 1 second.

[0052] In the device or system of the present disclosure, a pump (flow rate variable unit) can be attached at any location. The pump can be, for example, a syringe pump, a plunger pump, a piston pump, or a roller pump. The flow rate and pressure can be adjusted by the pump. In one embodiment, the pump that flows the first fluid and / or the second fluid is a plunger pump. In the present disclosure, a plunger pump may be preferred because it may be preferable to maintain a constant flow rate for a long period of time to examine various conditions. In one embodiment, the pump in a device or system of the present disclosure is capable of providing a flow rate of about 2 mL / min or more, about 5 mL / min or more, about 10 mL / min or more, about 20 mL / min or more, about 30 mL / min or more, about 40 mL / min or more, about 50 mL / min or more, about 60 mL / min or more, about 70 mL / min or more, about 80 mL / min or more, about 90 mL / min or more, about 100 mL / min or more, about 200 mL / min or more, about 300 mL / min or more, about 400 mL / min or more, about 500 mL / min or more, about 600 mL / min or more, about 700 mL / min or more, about 800 mL / min or more, about 900 mL / min or more, about 1000 mL / min or more, about 2000 mL / min or more, about 3000 mL / min or more, about 4000 mL / min or more, or about 5000 mL / min or more. Such high flow rates cannot be achieved with many research pumps. In one embodiment, the back pressure applied to the pumps that flow the first fluid and / or the second fluid is about 0.2 MPa or more, about 0.5 MPa or more, about 1 MPa or more, about 1.5 MPa or more, about 2 MPa or more, about 3 MPa or more, about 4 MPa or more, about 5 MPa or more, about 6 MPa or more, about 7 MPa or more, about 8 MPa or more, about 9 MPa or more, about 10 MPa or more, about 15 MPa or more, about 20 MPa or more, about 25 MPa or more, about 30 MPa or more, about 35 MPa or more, about 40 MPa or more, about 45 MPa or more, or about 50 MPa or more. In one embodiment, the pumps in the device or system of the present disclosure are capable of withstanding such high flow rates and high pressures, and a pump for high-performance liquid chromatography can be used. In the present disclosure, the fluid does not need to flow through narrow channels with high resistance or channels containing fillers, but it may be possible to flow the fluid through long channels at high flow rates, which may increase the back pressure on the pump.This is the first attempt to inject or collect small amounts of sample (e.g., 1 mL or less of sample, 1 mg or less of component) using such a high flow rate and / or high pressure pump, and the configurations of the method, system, and device disclosed herein for this purpose have not been seen before. Depending on the desired lipid particles, there may be cases where the particles cannot be successfully prepared unless they are prepared under high back pressure, and therefore the method, system, and device disclosed herein may be suitable for preparing such lipid particles.

[0053] In the device or system of the present disclosure, any pipe can be used for transporting liquid, and the material of the pipe can be determined taking into consideration, for example, insulation properties (thermal conductivity), heat resistance, chemical resistance, or airtightness. Examples of materials for the liquid transport tube (which is also the channel wall) and / or the connecting portions for forming the channel include, but are not limited to, glass, thermoplastics (e.g., polyvinyl chloride containing a plasticizer), thermoplastic elastomers (e.g., polyvinyl chloride without a plasticizer, a copolymer of styrene-ethylene-butylene and silicone oil, or a polypropylene-based plastic containing USP petroleum, polyether ether ketone (PEEK) (an aromatic polyether ketone in which benzene rings are bonded with ether groups and ketone groups), thermosetting rubber (e.g., a siloxane polymer containing amorphous silica), or thermally coagulable fluororubber. The channel may have any inner diameter, for example, about 0.1 mm, about 0.15 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, or about 1 mm.

[0054] In the device or system of the present disclosure, the confluence may include a mixer. An appropriate mixer may be selected taking into consideration factors such as the composition of the solution, flow rate, and pressure, and a T-shaped mixer, a mixer including a microchannel, a static mixer, or the like may be used. The mixer may be installed downstream of the confluence (e.g., a static mixer).

[0055] In one embodiment, the first flow path and the second flow path each include a plurality of divided flow paths, and the confluence section includes a plurality of split flow sections where the divided flow paths of the first flow path and the divided flow paths of the second flow path join together. In one embodiment, the divided flow paths are formed by branching the first flow path and / or the second flow path. In one embodiment, the divided flow paths downstream of the split flow sections join together in the third flow path. By using such divided flow paths, it is possible to scale up the system while maintaining the same conditions.

[0056] In one embodiment, the third or sixth flow path comprises a collection section, and a collection device is attached to the collection section. In one embodiment, the control unit further controls the time of collection by the collection device. In one embodiment, the collection section is located in the middle of the third or sixth flow path. In one embodiment, the collection device comprises a valve. In one embodiment, the valve is a sliding solenoid valve. In one embodiment, the device or system further comprises a receiver that receives fractions removed from the collection device. In one embodiment, the receiver includes multiple compartments that receive multiple fractions. In one embodiment, the device or system comprises a detector downstream of the junction.

[0057] The device or system of the present disclosure will be described with reference to specific embodiments shown in Figures 1 to 3. As shown in Figure 1A, the device or system of the present disclosure includes at least a first flow path (104a) and a second flow path (104b), each including an injection section (102). Pumps can be used to supply fluid to the first flow path (104a) and the second flow path (104b), and the flow rates of each can be independently set. An autosampler (101) is attached to the injection section (102), allowing automated formation of composites under various conditions. The first flow path (104a) and the second flow path (104b) are connected to a junction (105), from which a third flow path (104c) extends. After mixing of the first and second components, a fifth flow path (104d) may be further joined at an additional junction (105), in which case the composite flows through a sixth flow path (104e). After the first and second components are mixed, a fourth flow path (which may be 104d) for carrying the third component may be further joined at an additional joining section (105). If it is desired to collect only a specific fraction, the fraction is extracted using a collection device (107) (which may include a valve (108)) attached to the third or sixth flow path and collected in a receiving section (109). The control section (106) controls the injection sections (102) on at least the first flow path (104a) and the second flow path (104b) and issues instructions regarding the timing of sample injection so that the first and second components are simultaneously present and contact (mix) with each other at the joining section (105) where these flow paths join. The control section (106) may also control the autosampler (101) to automatically fill the injection sections (102) sequentially with (different) samples. The control unit (106) may control the collection device (107) to collect only a specific fraction (including the compound) at a specific timing. As shown in FIG. 2, the device or system of the present disclosure may have a detector installed in the third or sixth flow path downstream of the junction or additional junction. The detector can detect the first component, the second component, and / or the compound. The device or system of the present disclosure may further include a control unit (computer, 110) that controls the fluid (e.g., flow rate) flowing through the flow path, and can simultaneously control the autosampler and detector.

[0058] As shown in FIG. 1B, the device or system of the present disclosure may also be configured to include a split channel (104c') and / or a split flow section (105').

[0059] The system (lipid particle production system) of the present disclosure may have a control unit 30 as shown in Fig. 3A. The control unit 30 has a control unit 31 and a detection unit 32. The control unit 31 and the detection unit 32 are connected to each other so that they can communicate with each other. The above control may be executable by hardware (e.g., a dedicated circuit) alone, or may be executed by having a CPU execute a program.

[0060] In one aspect, the system (lipid particle production system) of the present disclosure may have a control unit 30 as shown in Figure 3B. The control unit 30 has a control unit 31, a detection unit 32, a recording unit 33, and a calculation unit 34. The control unit 31, the detection unit 32, the recording unit 33, and the calculation unit 34 are connected to each other so that they can communicate with each other. The above control may be executable by hardware (e.g., a dedicated circuit) alone, or may be executed by having a CPU execute a program.

[0061] The detector 45 detects the component concentration or the particle size of the lipid particles produced. The data acquired by the detector 45 is transmitted to the detection unit 32 and stored in the recording unit 33. The calculation unit 34 calculates the operating conditions of the system, such as the flow rate to be evaluated next, based on the information stored in the recording unit 33 (together with input from the input unit 41 as necessary).

[0062] The control unit 31 is composed of a CPU (Central Processing Unit), ROM (Read Only Memory), RAM (Random Access Memory), and drive circuits for various actuators included in the liposome-producing apparatus. The ROM stores various programs such as a BIOS (Basic Input / Output System), an OS (Operating System), various drivers, and various applications. The detection unit 32 is composed of detection circuits for various sensors included in the liposome-producing apparatus (e.g., temperature sensors, pressure sensors, and detectors).

[0063] The control unit 30 is communicatively connected to an input unit 41, a display unit 42, a storage unit 43, and an interface 44. The interface 44 enables data transmission and reception between the control unit 30 and an external device. The control unit 30 is connected to, for example, a general-purpose computer (a so-called personal computer) via the interface 44.

[0064] The input unit 41 accepts input from a user. The input unit 41 is configured, for example, by a keyboard, a mouse, or a touch panel. The display unit 42 is configured, for example, by a display such as an LCD (Liquid Crystal Display) or an ELD (Electro Luminescence Display). Note that when the input unit 41 and the display unit 42 are configured by a touch panel, the input unit 41 and the display unit 42 are integrated.

[0065] The storage unit 43 is composed of a non-volatile memory such as a hard disk, and stores programs and data related to various controls (for example, data input to the control unit 30 from the input unit 41). The control unit 31 controls the liquid supply pipes and the pumps 3a, 3b, and 3c based on information output from the calculation unit 34.

[0066] The control unit 31 controls at least one of the thermostatic bath, the liquid supply pipe, and the pumps 3 a, 3 b, and 3 c based on at least one of the data input from the input unit 41 to the control unit 30, the output signals of the temperature sensor and the pressure sensor input to the detection unit 32, and the information output from the calculation unit 34. The length of the liquid supply pipe can be controlled, for example, by switching the flow path.

[0067] In one aspect, the present disclosure provides a program that causes an apparatus including a processor to execute a method for injecting multiple components into multiple flow paths connected to a junction, which may be used in the methods, apparatuses, or systems described herein. The method executed by the program can cause the processor to perform processes including receiving information about a first flow path and information about a second flow path, determining a first time for injecting a first component into the first flow path and a second time for injecting a second component into the second flow path based on the information about the first flow path and the information about the second flow path, where the first time and the second time are times such that the first component injected into the first flow path and the second component injected into the second flow path reach the junction in a time to be mixed, and instructing the injection device to inject the first component into the first flow path at the first time and instructing the injection device to inject the second component into the second flow path at the second time. In one embodiment, the processing further includes receiving information about a third flow path downstream of the junction, determining a third time to withdraw a fraction from the third flow path based on the information about the third flow path, and instructing the collection device to withdraw the fraction from the third flow path at the third time.

[0068] Any control by the control unit described herein may be performed by a method coded by a program. In one aspect, the present disclosure also provides a recording medium having the program described herein recorded or stored thereon.

[0069] (Formulations) The present disclosure also provides a compound produced by the method of the present disclosure or a formulation containing the compound. The compound produced by the method of the present disclosure or a formulation containing the compound can be used for any purpose, such as pharmaceutical applications, food, cosmetics, agriculture, imaging, etc., and may be used as an insecticide, herbicide, cosmetic agent, fragrance, food additive, flavoring, imaging agent, dye, fluorescent marker, hair growth agent, moisturizer, pigment, skin whitening agent, pigment, X-ray contrast agent, ultrasound diagnostic agent, radioisotope-labeled nuclear medicine diagnostic agent, nuclear magnetic resonance diagnostic agent, etc. The formulation may also contain additives such as an osmotic pressure adjuster, stabilizer, antioxidant, pH adjuster, etc.

[0070] Examples of stabilizers include, but are not limited to, sugars such as glycerol, mannitol, sorbitol, lactose, or sucrose, and sterols such as cholesterol.

[0071] The antioxidant is not particularly limited, but examples thereof include ascorbic acid, uric acid, and tocopherol homologues (e.g., vitamin E). Tocopherol exists in four isomers, α, β, γ, and δ, and any of these can be used in the present invention.

[0072] The pH adjuster may include any basic or acidic compound, such as sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium malate, sodium succinate, and the like.

[0073] Examples of other additives include pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), PBS, biodegradable polymers, serum-free media, surfactants acceptable as pharmaceutical additives, and buffer solutions at physiological pH.

[0074] (General Techniques) Analytical techniques, chemical techniques, and pharmaceutical techniques used herein are well known and commonly used in the art, and are described, for example, in (Gregory Gregoriadis, Liposome Technology: Liposome Preparation and Related Techniques, September 12, 2006 CRC Press, ISBN 9780849388217), the relevant portions of which (possibly in their entirety) are incorporated herein by reference.

[0075] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within a range" of "two values" is specified, the range includes the two values ​​themselves.

[0076] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0077] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0078] (Example 1: Construction of a System) A system was constructed as shown in Figure 1A. The autosampler (101) and high-speed automatic valve (102) were controlled by a PLC (106) so that nucleic acids (flowing through the channel 104a) and lipids (flowing through the channel 104b) were mixed in a mixer (105). A high-speed dispenser (107) was used to precisely separate small amounts of lipid particles flowing at high speed. A solenoid slider valve (108) was attached to the high-speed dispenser to prevent small amounts of lipid particles from being stirred or diluted within the valve. The PLC also controlled the high-speed dispenser (107), and the process from injecting nucleic acids and lipids to collecting lipid particles was fully automated.

[0079] As shown in Figure 2, an in-line detector (111) is also installed to quantify nucleic acid (lipid) concentrations in real time. The LC system is controlled by a computer (which can also be a control unit) (110). The HPLC device (controller portion) and the PLC communicated directly with each other. The computer ran the HPLC control application, and signal exchange was carried out by the HPLC device itself. For example, it is possible to: 1) output a signal to the PLC when autosampler 1 is ready, 2) output a signal to the PLC when autosampler 2 is ready, and 3) output a time program start signal to the HPLC device when signals 1) and 2) are aligned.

[0080] In the method and system of the present disclosure, different samples must be mixed in a timely manner, and conditions for achieving this were investigated. A test was conducted on the mixing of a nucleic acid sample (detected by absorbance at OD260) and a lipid sample (detected by absorbance at OD600), which are distinguishable due to their different absorbance wavelengths. These samples were injected into separate flow paths, and the resulting mixtures were combined to obtain consecutive fractions, which were then measured for absorbance.

[0081] A valve was attached to a flow path with a flow rate of 45 mL / min, through which 100 μL of a nucleic acid sample was injected, and a valve was attached to another flow path with a flow rate of 15 mL / min, through which 200 μL of a lipid sample was injected, and these flow paths were joined in a mixer. Mixing was tested by replacing the piping from the valve to the mixer under the condition that a sample injection signal was sent to the two valves simultaneously.

[0082] The results are shown in Figure 4. When two flow paths were fitted with tubing of the same inner diameter and length, the timing at which each sample entered the mixer differed due to the difference in flow rate between the flow paths. As a result, the nucleic acids and lipids showed maximum concentrations in different fractions (Figure 4A). Next, when a tubing with an inner diameter of 0.381 mm and a length of 76 cm (87 μL) was added to the flow path for injecting the nucleic acid sample, the fractions showing the maximum concentrations of nucleic acids and lipids became closer (Figure 4B). Furthermore, when the tubing added to the flow path for injecting the nucleic acid sample was switched to one with an inner diameter of 0.381 mm and a length of 134 cm (153 μL), the nucleic acids were found to be at their maximum concentration in the fraction containing a sufficiently high concentration of lipids (Figure 4C). When a pipe with an inner diameter of 0.381 mm and a length of 76 cm (87 μL) was added to the channel for injecting the nucleic acid sample, the pipe from the valve of the channel for injecting the lipid sample to the mixer was changed to a smaller volume (−10 μL) pipe with an inner diameter of 0.18 mm and a length of 6 cm. Even under these conditions, nucleic acids were found to be present at the highest concentration in fractions containing sufficiently high concentrations of lipids (Figure 4D).

[0083] Next, we attached pipes with the same inner diameter and length to the two flow paths from the valve to the mixer, and attempted to properly mix the two components by controlling the injection timing. A high-speed automatic valve (VF-02: From) was used.

[0084] The results are shown in Figure 5. When sample injection signals were sent to two valves simultaneously, the timing at which each sample entered the mixer differed due to the difference in flow rate between the channels. As a result, the nucleic acids and lipids showed maximum concentrations in different fractions (Figure 5A). Next, when the injection timing of the nucleic acid sample was delayed by 100 msec from the injection timing of the lipid sample, the nucleic acids were found to be at their maximum concentration in the fraction containing a sufficiently high concentration of lipids (Figure 5B). Note that the decrease in lipid concentration in the seventh fraction is likely due to experimental error caused by pressure fluctuations resulting from the delayed valve switching operation, which pushed back the fluid in the lipid channel.

[0085] Such considerations can facilitate the establishment of conditions for achieving proper mixing between the components.

[0086] (Example 3: Study of valve) In the device configuration of Example 1, the influence of the valve on the injection of the sample and the recovery of the lipid particles was studied.

[0087] The conditions used in the test were as follows: Nucleic acid sample: 1.28 mg / mL nucleic acid (in water), injection volume: 200 μL. Lipid sample: Lipid composition: DOTAP:DPPC:Cholesterol:DSPE-MPEG2K = 50:19.5:30:0.5, lipid concentration: 50 mg / mL (in ethanol), injection volume: 200 μL. Flow rate (nucleic acid flow path: 45 mL / min, lipid flow path: 15 mL / min), N / P ratio = 3.0. Piping from the nucleic acid sample injection port to the mixer: inner diameter 0.381 mm, length 76 cm. Piping from the lipid sample injection port to the mixer: inner diameter 0.18 mm, length 6 cm. Flow rate for the flow path for water for secondary dilution: 23.3 mL / min. Inner diameter of the flow path after secondary dilution: 0.5 mm. Volume of obtained fractions: 80 μL per fraction. - Time interval until obtaining the next fraction: 0 ms (this is the setting value on the device. In reality, it is thought that a certain amount of time interval occurs.) - Autosampler settings (set on the PC with the HPLC control application): Input items: sample number, sample intake / injection speed, injection amount (sample amount + excess amount), amount of air injected before and after the sample, needle + injection port washing settings before and after injection, request signal output before sample injection, sample injection start signal input.

[0088] The autosampler, valves, and dispensers (collection devices) were controlled according to a program as follows: 1) The two autosamplers (master and slave) each sent an injection-ready signal to the PLC. 2) When both autosamplers sent the injection-ready signals, the PLC sent a time program start signal to the autosampler (the master side was used for the time program). 3) The pump was started according to the master side time program. 4) Using the time program, the autosampler sent a dispenser program start signal to the PLC. 5) The PLC sent a program start signal to the dispenser. 6) The dispenser program started (after cleaning the fractionation port, it moved to the fractionation start position (waiting for the fractionation start signal)). 7) After the pump reached the set flow rate (time setting), the autosampler (master side) sent an injection request signal to the PLC. 8) The PLC sent injection signals to the two valves. Simultaneously with this injection signal, a signal to start fraction collection and a signal to switch from the waste liquid side to the fraction collection side are sent to the dispenser. 9) Fraction collection is performed by the dispenser (the number of fractions and fraction volume per well / 96-well plate are preset in the dispenser program.) 10) After fraction collection, the dispenser moves to the washing position, washes the flow path, and then moves to the next fraction collection start position.

[0089] The following three valve conditions were considered: ・0.8 s + DV: Low-speed automatic valve (FCV-12AH: Shimadzu) + diaphragm valve (MTV-3-1: Takasago Electric) ・0.1 s + DV: High-speed automatic valve (VF-02: From) + diaphragm valve ・0.1 s + SV: High-speed automatic valve + slider valve (MTV-3SL: Takasago Electric)

[0090] The fractions obtained under each condition were examined for nucleic acid concentration (detected by absorbance at OD260), lipid concentration (detected by absorbance at OD600), and lipid particle size distribution (measured by dynamic light scattering).

[0091] (Results) Regarding nucleic acid concentration (Figure 6A), with 0.1s + SV, the number of fractions with intermediate concentrations before reaching the peak concentration was small, and the peak concentration plateaued over three fractions, suggesting that these fractions had the same effect as when the same nucleic acid concentration as the injected sample was continuously flowed. With 0.1s + DV, the peak concentration was slightly lower than with 0.1s + SV, and the number of fractions with intermediate concentrations below the peak concentration increased, indicating that a large portion of the nucleic acid concentration in the injected sample was not fully reflected due to dilution. With 0.8s + DV, the peak concentration was low, and it was observed that the nucleic acid concentration was lower overall than that of the injected sample due to dilution.

[0092] Regarding lipid concentration (Fig. 6B), with 0.1s + SV, the number of fractions with intermediate concentrations before reaching the peak concentration was small, and the peak concentration plateaued over five fractions, suggesting that these fractions had the same effect as continuing to flow the same lipid concentration as the injected sample. With 0.1s + DV, the peak concentration was slightly lower than with 0.1s + SV, and the number of fractions with intermediate concentrations below the peak concentration increased, indicating that dilution did not adequately reflect the lipid concentration in the injected sample. With 0.8s + DV, the peak concentration was low, and it was observed that the lipid concentration was lower overall than the injected sample due to dilution.

[0093] Regarding particle size distribution (Figure 7 and the table below), the average particle size of the three fractions that gave the nucleic acid peak concentration was almost the same. With 0.1s+DV, the average particle size differed between the fraction that gave the nucleic acid peak concentration and the fractions before and after it, which is thought to be due to the nucleic acid / lipid ratio (A260 / A600) not being constant. With 0.8s+DV, the three fractions that gave the highest nucleic acid peak concentration had small average particle sizes, possibly due to the low peak nucleic acid concentration caused by dilution, and large PDI and coarse particles were observed due to uneven mixing.

[0094]

[0095] (Example 4: Examination of mass production) It was examined whether the manufacturing conditions set for a small sample could be scaled up to mass production. The conditions and results are summarized in the table below.

[0096]

[0097] It was confirmed that larger-scale production is possible using the same conditions as when using a small amount of sample. The performance of the obtained particles was similar under all conditions, confirming that scale-up is still possible even when reactions such as particle formation are involved, not just simple mixing.

[0098] The main configurations of the small-scale automatic manufacturing equipment and mass production equipment were as follows:

[0099]

[0100] (Example 5: Mixing of three components) A valve was further attached to the flow path for the water used for secondary dilution in Example 2, allowing an acidic polymer (poly-γ-glutamic acid) to be injected into this flow path, and a test was conducted to determine whether mixing of three components, i.e., nucleic acid, lipid, and acidic polymer, in one device was possible.

[0101] The flow conditions for each component are shown in the table below. In condition 1, similar to the device configuration of Example 2, the flow paths were configured so that the flow path for injecting the nucleic acid sample and the flow path for injecting the lipid sample joined together, and then the flow path for injecting the acidic polymer joined together. In condition 2, the flow paths were configured so that the three flow paths for injecting the nucleic acid sample, the flow path for injecting the lipid sample, and the flow path for injecting the acidic polymer joined together at one point. For each condition, fractions with high OD260 absorbance, which indicates nucleic acid concentration, were obtained, and the particle size distribution and z-potential of those fractions were measured.

[0102]

[0103] The results are shown below. LNPs were formed by mixing nucleic acids, lipids, and acidic polymers from their respective channels, and the z-potential measurements indicated that the prepared LNPs had nucleic acids and lipids in the core and acidic polymers in the shell. It can be seen that the method and device disclosed herein can be used to examine particle preparation conditions with small amounts, even in particle formation requiring the mixing of three or more components.

[0104]

[0105] (Example 6: Three-component mixing, four pumps) The channel downstream of the confluence (1) between the channel for nucleic acid (pump A) and the channel for lipid (pump B) was joined with a channel for ethanol (pump D) equipped with a valve for injecting cationic lipid (DOTAP) (confluence 2), and further downstream, the channel was joined with a channel for dilution (pump C) (confluence 3) (see Figure 8). With this device configuration, we tested whether it was possible to mix three components, nucleic acid and two types of lipids, on a single device.

[0106] The flow conditions for each component are shown in the table below. Under condition 2, the same amount of ethanol solvent was injected instead of the cationic lipid. For each condition, fractions with high OD260 absorbance, which indicates nucleic acid concentration, were collected, and the particle size distribution and z-potential of the fractions were measured.

[0107]

[0108] The results are shown below. Comparing conditions 1 and 2, it can be seen that the addition of cationic lipid as an additional component positively charged the surface of the LNP. It can be seen that the method and apparatus of the present disclosure can examine particle production conditions with small amounts, even in particle formation that requires the sequential mixing of helper lipids that modify the surface layer of the LNP.

[0109]

[0110] (Example 7: Scale-up by parallel mixing) When forming lipid particles, the particle size tends to become smaller as the flow rate (linear flow rate) increases. In the present disclosure, which assumes a high flow rate, a method of decreasing the linear flow rate while maintaining a high flow rate was investigated so that lipid particles of various particle sizes can be formed.

[0111] The device configuration of Example 2 was modified so that the channel for injecting the nucleic acid sample and the channel for injecting the lipid sample were each branched into two divided channels, the two divided channels for injecting the nucleic acid sample and the two divided channels for injecting the lipid sample were merged at two divided flow sections, and the two flows from the two divided flow sections were merged at a junction, and then further merged with a channel carrying water for secondary dilution (see Figure 1B).

[0112] Compared to the device configuration in Example 2, piping of the same inner diameter and length was used for each divided channel. Tests were performed with total flow rates of 30 mL / min (nucleic acid: 16.2 mL / min, lipid: 5.4 mL / min, secondary dilution: 8.4 mL / min) and 60 mL / min (nucleic acid: 32.4 mL / min, lipid: 10.8 mL / min, secondary dilution: 16.8 mL / min).

[0113] The results are shown in the table below, comparing the results with a non-branched flow channel configuration. By using a split flow channel, it was possible to increase the particle size of the LNPs while maintaining a high flow rate. Similarly, by further increasing the number of branches, it may be possible to further mass-produce the LNPs while maintaining the same particle formation conditions.

[0114]

[0115] (Example 8: Installation of a Thermostatic Bath) A thermostatic bath (Shimadzu Corporation CTO-20AC) was installed in the section of the channel for injecting the nucleic acid sample and the channel for injecting the lipid sample, up to the point just before their confluence, and LNPs were formed under heating conditions at 60°C. The device configuration was the same as in Example 2. Nucleic acid sample: 1 mg / mL nucleic acid (in water), injection volume 0.1 mL. Channel for injecting the nucleic acid sample: flow rate (water) 15 mL / min. Lipid sample: 23 mg / mL lipid for LNP formation (in ethanol), injection volume 0.2 mL. Lipid composition (DOTAP:DPPC:DSPE-Glu:Cholesterol:DSPE-MPEG2K = 20:10:10:59:1) (this lipid sample undergoes lipid precipitation when stored at room temperature in normal ethanol). Channel for injecting the lipid sample: flow rate (ethanol) 45 mL / min. Flow rate for the water flow channel for secondary dilution: 23.3 mL / min.

[0116] The Z-average of the LNPs was 72.5 nm and the PDI was 0.075, confirming that good LNPs could be formed even when poorly soluble lipids were used.

[0117] (Note) As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted solely by the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2023-107138, filed with the Japan Patent Office on June 29, 2023, the contents of which are incorporated by reference in their entirety into this application.

[0118] According to the present disclosure, suitable reaction conditions can be determined for production with a small consumption of materials.

Claims

1. A method for producing a formulation containing a first component and a second component, comprising the steps of: preparing a series of flow paths, in which a first flow path through which a first fluid flows at a first flow rate and a second flow path through which a second fluid flows at a second flow rate, and which join at a junction to form a third flow path through which a third fluid flows at a third flow rate; injecting a sample containing the first component into the first flow path at a first time; and injecting a sample containing the second component into the second flow path at a second time, wherein the first time and the second time are set so that the first and second components reach the junction at the timing when they are mixed.

2. The method of claim 1, wherein said first component is injected into a middle portion of said first flow path and said second component is injected into a middle portion of said second flow path.

3. The method of claim 1 or 2, wherein the third time is set based on the first time and the second time.

4. The method of any one of claims 1 to 3, wherein the first flow rate or the second flow rate is greater than about 2.4 mL / min.

5. The method according to any one of claims 1 to 4, wherein the series of flow paths includes a fourth flow path through which a sample containing a third component flows, and the method further includes the step of injecting the third component into the fourth flow path at a third time, wherein the first time, the second time, and the third time are set so that the first component, the second component, and the third component reach the junction at the timing at which they are mixed.

6. The method of claim 5, wherein said third flow path and said fourth flow path join at an additional junction downstream of said junction.

7. The method according to claim 5 or 6, wherein the first flow path, the second flow path and the fourth flow path join at the junction.

8. The method according to any one of claims 1 to 7, further comprising the step of detecting a confluence state of the first component and the second component downstream of the confluence.

9. The method of claim 8, wherein the confluence state is detected as a state selected from the group consisting of the concentration of at least one of the first component and the second component, the particle size of particles in the flow, and the state of chemical bonding between the first component and the second component.

10. A method according to any one of claims 1 to 9, wherein in the step of injecting a sample containing the first component into the first flow path, the first flow path is in a liquid-flowing state, and in the step of injecting a sample containing the second component into the second flow path, the second flow path is in a liquid-flowing state.

11. A method according to any one of claims 1 to 10, wherein in the step of injecting a sample containing the first component into the first flow path and the step of injecting a sample containing the second component into the second flow path, the retention times of the sample containing the first component and the sample containing the second component after being filled in the sample injection section until being injected into the flow paths are independently within one minute.

12. The method according to any one of claims 1 to 11, wherein the time from when the first component and the second component are injected to when they reach the confluence is independently within 2 seconds.

13. A method according to any one of claims 1 to 12, wherein the step of injecting the first component into the first flow path injects 1000 μg or less of the first component, and the step of injecting the second component into the second flow path injects 1000 μg or less of the second component.

14. The method of any one of claims 1 to 13, wherein a fourth fluid flowing through a fifth flow path is joined with the third fluid at an additional junction to form a sixth flow path for carrying the fifth fluid.

15. The method according to any one of claims 1 to 14, wherein the confluence comprises a T-mixer, a microchannel device or a static mixer.

16. The method of any one of claims 1 to 15, wherein the third flow path comprises a mixer.

17. The method of claim 16, wherein the mixer on the third flow path is a static mixer.

18. A method according to any one of claims 1 to 17, wherein the first flow path and the second flow path each include a plurality of divided flow paths, and the junction portion includes a plurality of partial flow portions where the divided flow paths of the first flow path and the divided flow paths of the second flow path join together.

19. The method of claim 18, wherein the first flow path and / or the second flow path branch to form the divided flow path.

20. The method according to claim 18 or 19, wherein in the third flow path, the divided flow paths downstream of the split flow section join together.

21. The method according to any one of claims 1 to 20, further comprising the step of withdrawing a fraction containing a composite of the first component and the second component from a collection portion of the third flow path at a third time.

22. The method of claim 21, wherein the collection portion is located in a middle portion of the third flow path.

23. The method of claim 21 or 22, further comprising distributing said fraction into a receptor.

24. The method of claim 23, wherein said distributing step comprises distributing a plurality of said fractions into said receptacle containing a plurality of compartments.

25. The method of claim 24, further comprising the step of examining a plurality of said fractions.

26. The method according to any one of claims 21 to 25, wherein the time at which the fraction is removed from the collection section is controlled based on the time and flow rate at which the component is injected into the flow path.

27. An apparatus for injecting multiple samples into multiple flow paths at a timing when a first component and a second component are mixed, comprising: a first flow path for flowing a first fluid; a first injection unit for injecting a sample containing the first component into the first flow path; a second flow path for flowing a second fluid; a second injection unit for injecting a sample containing the second component into the second flow path; a junction unit where the first flow path and the second flow path are joined; a third flow path extending from the junction unit for flowing a third fluid; and a control unit, wherein the control unit is configured to control the time when the first injection unit injects the first component into the first flow path and the time when the second injection unit injects the second component into the second flow path.

28. The apparatus of claim 27, wherein the first inlet is positioned in a middle portion of the first flow path and the second inlet is positioned in a middle portion of the second flow path.

29. The device of claim 27 or 28, wherein the first injection section is a valve capable of injecting a first component into the first flow path within 5 seconds, and the second injection section is a valve capable of injecting a second component into the second flow path within 5 seconds.

30. The device of any one of claims 27 to 29, wherein at least one of the first injection section and the second injection section is capable of switching flow paths within about 500 ms.

31. The device according to any one of claims 27 to 30, further comprising: a fourth flow path for flowing a third fluid; and a third injection unit for injecting a sample containing a third component into the fourth flow path, wherein the series of flow paths includes the fourth flow path, and the control unit is configured to control the time during which the first injection unit injects a first component into the first flow path, the time during which the second injection unit injects a second component into the second flow path, and the time during which the third injection unit injects a third component into the fourth flow path.

32. An apparatus as claimed in any one of claims 27 to 31, including a detector downstream of the junction.

33. An apparatus according to any one of claims 27 to 32, wherein the first injection section includes a valve with a sample loop having a volume of 5 mL or less, and the second injection section includes a valve with a sample loop having a volume of 5 mL or less.

34. The apparatus of any one of claims 27 to 33, wherein the pumps for flowing the first fluid and the second fluid are plunger pumps.

35. An apparatus according to any one of claims 27 to 34, wherein a back pressure applied to the pumps for flowing the first fluid and the second fluid is 1 MPa or more.

36. The apparatus of any one of claims 27 to 35, further comprising: a fourth flow path for carrying a fourth fluid; an additional junction where the fourth flow path and the third flow path join; and a fifth flow path for carrying a fifth fluid extending from the additional junction.

37. The apparatus of any one of claims 27 to 36, wherein the third flow path comprises a collection portion, the collection portion further comprising a collection device.

38. An apparatus according to any one of claims 27 to 37, further comprising a thermostatic chamber that maintains the first, second and third flow paths, the first and second injection sections, and at least a portion of the confluence section at a constant temperature.

39. The apparatus of claim 37 or 38, wherein the control unit further controls the time of collection of the collection device.

40. The apparatus of claim 39, wherein the control unit controls at least one of the sampling start time and the sampling time interval of the sampling device.

41. The device of any one of claims 38 to 40, wherein the collection portion is located in a middle portion of the third flow path.

42. Apparatus according to any one of claims 38 to 41, wherein the collection device comprises a valve.

43. The apparatus of claim 42, wherein the valve is a sliding solenoid valve.

44. The apparatus of any one of claims 38 to 43, further comprising a receiver for receiving a fraction removed from the collection device.

45. The device of claim 44, wherein the receptacle includes multiple compartments for receiving multiple fractions.

46. ​​A program for causing an apparatus having a processor to execute a method for injecting multiple components into multiple flow paths connected to a junction, the method including: receiving information regarding a first flow path and information regarding a second flow path; determining a first time for injecting a sample containing a first component into the first flow path and a second time for injecting a sample containing a second component into the second flow path based on the information regarding the first flow path and the information regarding the second flow path, wherein the first time and the second time are times such that the first component injected into the first flow path and the second component injected into the second flow path reach the junction at a timing at which they are mixed; instructing an injection device to inject the sample containing the first component into the first flow path at the first time; and instructing the injection device to inject the sample containing the second component into the second flow path at the second time.

47. The program of claim 46, wherein the method further comprises: receiving information regarding a third flow path downstream of the junction; determining a third time for removing a fraction from the third flow path based on information regarding the third flow path; and instructing a collection device to remove the fraction from the third flow path at the third time.

48. A recording medium storing a program for causing an apparatus having a processor to execute a method for injecting multiple components into multiple flow paths connected to a junction, the method including: receiving information regarding a first flow path and information regarding a second flow path; determining a first time for injecting a sample containing a first component into the first flow path and a second time for injecting a sample containing a second component into the second flow path based on the information regarding the first flow path and the information regarding the second flow path, the first time and the second time being times such that the first component injected into the first flow path and the second component injected into the second flow path reach the junction at a timing at which they are mixed; instructing an injection device to inject the sample containing the first component into the first flow path at the first time; and instructing the injection device to inject the sample containing the second component into the second flow path at the second time.

49. The recording medium of claim 48, wherein the method further comprises: receiving information regarding a third flow path downstream of the junction; determining a third time for removing a fraction from the third flow path based on information regarding the third flow path; and instructing a collection device to remove the fraction from the third flow path at the third time.

50. A method for screening production conditions for lipid particles loaded with drugs, comprising the steps of: preparing a series of flow paths, in which a first flow path through which a first fluid flows at a first flow rate and a second flow path through which a second fluid flows at a second flow rate join at a junction to form a third flow path through which a third fluid flows at a third flow rate; (1) injecting a sample containing a candidate lipid into the first flow path at a first time; (2) injecting a sample containing a candidate drug into the second flow path at a second time; (3) extracting a fraction containing candidate lipid particles from a collection portion of the third flow path at a third time; and evaluating the candidate lipid particles, wherein the first time and the second time are set so that the candidate lipid and the candidate drug arrive at the junction simultaneously.

51. The method of claim 50, further comprising the steps of preparing a plurality of candidate lipid particles by repeating steps (1) to (3), comparing the plurality of candidate lipid particles, and selecting a hit lipid particle.

52. A method for producing the hit lipid particle described in claim 51 under production conditions set in the screening method described in claim 50.

53. A method for producing hit lipid particles as described in claim 52, wherein the production yield of the hit lipid particles is increased by increasing the frequency of performing steps (1) to (3).

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