System and method for recovering antigen in solvent

Abstract

A system (A) for recovering an antigen in a solvent, said system being provided with: charged particles that contain 3-70 mol% of a cationic lipid, 1-20 mol% of a PEG-containing lipid, and 20-50 mol% of cholesterol, the surface of the charged particles being modified with an antibody, the average zeta potential of the charged particles being-20-+ 15 mV; the mixing unit (4) is configured so that the charged particles are introduced into a solvent (C) in the mixing unit (4) and the charged particles and the solvent (C) are stirred, and the recovery unit (7) is configured so as to recover the charged particles mixed with the solvent (C), and the recovery unit (7) is an anionic adsorber provided with an adsorption carrier. A compound having an anionic functional group is fixed to the surface of the adsorption carrier.

Description

Technical Field

[0001] The invention relates to an antigen recovery system in a solvent and an antigen recovery method in a solvent. This application claims priority based on Japanese Patent Application No. 2022-128953 filed in Japan on August 12, 2022, the contents of which are hereby incorporated by reference. Background Art

[0002] As a method for effectively removing blood components (low-molecular compounds, proteins, nucleic acids, virus particles, bacterial cells, etc.) that may be harmful to health, a more effective and safer method is required. As the substances to be removed, for example, urea, creatinine, uric acid, β2 microglobulin, LDL cholesterol, abnormal antibodies in immune diseases, viruses, bacteria, fungi, cancer cells and other substances that are the cause of the disease can be listed. In addition to these substances, endotoxins, cytokines such as IL-6, heavy metal ions and the like can also be listed.

[0003] Methods for removing specific components from the blood include a method of administering a drug and removing the component based on the action mechanism of the drug and a method of directly removing the specific component from the blood, that is, direct removal technology. Examples of the former include the use of antibiotics against pathogenic bacteria and the use of antiviral agents against pathogenic viruses. An example of the latter direct removal technique is a magnetic separation method using antibody-modified magnetic beads.

[0004] In direct removal technology, blood is taken out of the body and treated to selectively remove specific components. The blood after treatment is returned to the body. Direct removal technology is relatively easy to develop because of its simple mechanism of action. In addition, it is expected that direct removal technology can achieve results in a relatively short time.

[0005] As a direct removal technique of a magnetic separation method using antibody-modified magnetic beads, the technique described in Non-Patent Document 1 is known. Prior art literature Non-patent literature

[0006] JHKang et al.,small,2015,11,No.42,5657-5666 Summary of the invention Problems to be solved by the invention

[0007] In the system for removing antigens from blood, biocompatibility is one of the important indicators. In conventional magnetic separation methods including the technology described in Non-Patent Document 1, magnetic beads made of inorganic substances are used. These inorganic substances are substances that do not originally exist in the body and are not metabolized in the body. The present disclosure provides systems and methods for effectively removing specific components from a solvent using a medium having higher biocompatibility than magnetic beads. Means for solving problems

[0008] In order to solve the above-mentioned problems, the present disclosure has the following aspects.

[0009] A system for recovering antigens in a solvent comprises charged particles, a mixing unit and a recovery unit, wherein the charged particles contain 3 to 70 mol% of a cationic lipid, 1 to 20 mol% of a PEG-containing lipid, 20 to 50 mol% of cholesterol and 0 to 65 mol% of a neutral lipid. The surface of the charged particle is modified with antibodies, The average zeta potential of the charged particles is -20 to +15 mV, The mixing section is configured such that the charged particles are put into a solvent and the charged particles and the solvent are stirred in the mixing section. The recovery unit is configured to recover the charged particles mixed with the solvent. The recovery unit is an anionic adsorber including an adsorption carrier having a compound having an anionic functional group fixed on the surface of the adsorption carrier.

[0010] A method for recovering antigens in a solvent, comprising: The step of adding charged particles into a solvent and stirring; and a step of recovering the charged particles mixed with the solvent, The charged particles contain 3 to 70 mol% of cationic lipids, 1 to 20 mol% of PEG-containing lipids, 20 to 50 mol% of cholesterol and 0 to 65 mol% of neutral lipids. The surface of the charged particle is modified with antibodies, The average zeta potential of the charged particles is -20 to +15 mV. The charged particles mixed with the solvent are recovered by electrostatic adsorption via an adsorption carrier having a compound having an anionic functional group fixed on the surface thereof. Effects of the Invention

[0011] According to the system and method for recovering antigens in a solvent according to the present disclosure, it is possible to effectively remove a predetermined component (antigen) in a solvent (eg, blood) using a medium with high biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of an example of an antigen recovery system in a solvent according to an embodiment of the present disclosure. Figure 2 Schematic diagram of charged particles used in an antigen recovery system in a solvent according to an embodiment of the present disclosure. Figure 3 Detailed description of the invention is a flow chart showing the steps of a method for recovering an antigen in a solvent according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0013] Hereinafter, the embodiments of the present disclosure will be described in detail with appropriate reference to the accompanying drawings. In the drawings used in the following description, in order to make the features easier to understand, the features are sometimes enlarged for convenience, and the dimensional ratios of the components are sometimes different from the actual ones. The materials, dimensions, etc. illustrated in the following description are examples, and the present disclosure is not limited thereto, and can be implemented after being appropriately changed within the scope of exerting the effects of the present disclosure.

[0014] (Antigen Retrieval System in Solvent) Figure 1 Schematic diagram of an example of an antigen recovery system in a solvent according to an embodiment of the present disclosure. Figure 2 Schematic diagram of charged particles used in an antigen recovery system in a solvent according to an embodiment of the present disclosure. Figure 3 Detailed description of the invention is a flow chart showing the steps of a method for recovering an antigen in a solvent according to an embodiment of the present disclosure.

[0015] Figure 1 The illustrated antigen recovery system A in a solvent includes charged particles B, a mixing unit 4 , and a recovery unit 7 . The charged particles B contain 3 to 70 mol% of cationic lipids, 1 to 20 mol% of PEG-containing lipids and 20 to 50 mol% of cholesterol. The surface of the charged particle B is modified with the antibody 25, and the average zeta potential of the charged particle B is -20 to +15 mV. The mixing section 4 is configured such that the charged particles B are put into a solvent C and the charged particles B and the solvent C are stirred in the mixing section 4 . The recovery unit 7 is configured to recover the charged particles B mixed with the solvent C. The recovery unit 7 is an anionic adsorber including an adsorption carrier having a compound having an anionic functional group fixed on the surface of the adsorption carrier.

[0016] The antigen recovery system A in a solvent further comprises a first tank 1 , a pump 6 , and a second tank 8 in addition to the essential components described above. The mixing unit 4 may include a charged particle introduction unit 2 and a mixer 3 . The antigen recovery system A in a solvent may further include flow paths 11 to 15 .

[0017] According to the antigen recovery system A in a solvent, antigens in a solvent can be removed efficiently using a medium having higher biocompatibility than that of the prior art.

[0018] <Charged Particles> Figure 2 Schematic diagram of charged particles used in an antigen recovery system in a solvent according to an embodiment of the present disclosure. The charged particle B is a liposome containing a cationic lipid 21 , a neutral lipid 22 , a PEG-containing lipid 24 , and an antibody 25 .

[0019] Cationic lipid 21 is a positively charged lipid. Examples of lipids that can be used as cationic lipid 21 include DOTAP (1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)), DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), DC-Chol (3-3-N-(N',N'-dimethylmethane)carbamoylcholesterol), and DMRIE (dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium). Among cationic lipids, dendrimers, DOSPER (1,3-dioleoyloxy-2-(6-carboxysperminyl)propionamide), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), etc. are not suitable as cationic lipids 21 (which are components of charged particles B) because of the strong electrostatic interaction between their positive charges and antibodies, which reduces the antibody activity.

[0020] The content of the cationic lipid 21 in the charged particles B is 3 to 70 mol%. If the content of the cationic lipid 21 is outside the above range, it is difficult to set the average zeta potential within a predetermined range described below. If the content of the cationic lipid 21 is outside the above range, the electrostatic interaction with the adsorption monomers provided in the anionic adsorber as the recovery unit 7 is insufficient, and the recovery efficiency is reduced. The content of the cationic lipid 21 in the charged particles B may be 10 to 60 mol %, or 25 to 55 mol %.

[0021] The types and contents of the components in the charged particles B (including cationic lipids, neutral lipids, PEG-containing lipids, cholesterol, etc.) can be identified and measured by methods such as high performance chromatography, thin layer chromatography, and mass spectrometry (LC-MS).

[0022] Neutral lipid 22 is a lipid that is neither positively nor negatively charged or a lipid that contains equal amounts of positive and negative charges. Examples of lipids that can be used as neutral lipid 22 include DSPC (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), HSPC (hydrogenated soybean phosphatidylcholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), and DOPE (dioleoylphosphatidylethanolamine).

[0023] The content of the neutral lipid 22 in the charged particles B is 0 to 65 mol%. If the content of the neutral lipid is outside the above range, it becomes difficult to adjust the liposomes and set the average zeta potential within a predetermined range described later. The content of the neutral lipid 22 in the charged particles B may be 0 to 30 mol %, or 0 to 25 mol %.

[0024] The PEG-containing lipid 24 is a lipid to which a predetermined PEG (polyethylene glycol) is covalently bonded.

[0025] As the PEG of the PEG-containing lipid 24, PEG having a molecular weight of 1000 to 6000 Da can be used. The molecular weight of PEG may be 1800 to 5500 Da, or 2000 to 3000 Da. More specifically, PEG may be PEG2000 or PEG5000.

[0026] As the PEG-containing lipid 24, DSPE-PEG (2000) maleimide, DSPE-PEG (5000) maleimide, DMG-PEG (1,2-dimyristoyl-rac-glycero-3-methoxypentadienyl alcohol), DSG-PEG (distearoyl-rac-glycero-polyethylene glycol), etc. can be used.

[0027] The content of the PEG-containing lipid 24 in the charged particles B is 1 to 20 mol%. If the content of the PEG-containing lipid 24 is outside the above range, it is difficult to set the average zeta potential within the specified range described below, similarly to the case of the cationic lipid. If the content of PEG-containing lipid 24 is outside the above range, when charged particles B are mixed and diffused with solvent C, the specificity of binding between the antigen in the solvent and the antibody presented on the surface of charged particles B decreases, and the efficiency of antigen recovery decreases. The content of the PEG-containing lipid 24 in the charged particles B may be 6 to 14 mol %, or 8 to 12 mol %.

[0028] The antibody 25 in the charged particle B is an antibody having a substance to be removed from the solvent as an antigen. The antibody 25 may be any antibody as long as it has a predetermined antigen specificity and can be used for modulation of the charged particle B. If the above conditions are met, the antibody 25 may lack a part of the basic structure of a general antibody, or may be partially replaced by a module having a function that the antibody does not originally have. As the antibody 25, an antibody in which amino acid substitution, insertion or deletion is artificially introduced into a wild-type antibody can be used.

[0029] As the antibody 25 in the charged particle B, a polyclonal antibody, a monoclonal antibody, an antibody produced by phage display, or the like can be used.

[0030] The content of antibody 25 in charged particle B is 5 to 1000 μg / mg (antibody mass / charged particle). When the antibody content is less than 5 μg / mg, sufficient antigen recovery efficiency cannot be obtained. When the antibody content exceeds 1000 μg / mg, the characteristics of charged particle B (stability of charged particle, antigen recovery efficiency) deteriorate. The content of the antibody 25 in the charged particles B may be 20 to 200 μg / mg, or 30 to 100 μg / mg.

[0031] The zeta potential is the potential difference between the sliding surface and a portion sufficiently away from the interface in the double electric layer. The zeta potential is a parameter that serves as an index of the dispersion state of particles in a colloidal liquid, etc. The average zeta potential of the charged particles in the present disclosure is defined as a parameter required for specifically removing antigens from a solvent.

[0032] The average zeta potential of the charged particles B is -20 to +15 mV. When the average zeta potential of the charged particles B is less than -20 mV, even if the antigen and the antibody bind, the recovery efficiency in the recovery unit 7 as an anion adsorber will decrease. When the average zeta potential of the charged particles B exceeds +15 mV, the binding of nonspecific antigens and antibodies due to electrostatic interaction is enhanced, and it becomes impossible to efficiently and specifically recover the antigen. The average zeta potential of the charged particle B may be -15 to +10 mV or -10 to +5 mV.

[0033] The average zeta potential of the charged particles B (and the average zeta potential of the anion adsorber described later) can be measured by a commonly used method using the Smoluchowski equation.

[0034] For example, when measuring the zeta potential of a predetermined liposome dispersion (HEPES 0.2 mM NaCl 3 mM pH 7.2), the measurement conditions are a measurement temperature of 25° C. and a liposome concentration of 0.05 to 0.4 mg / ml. In the zeta potential measurement setting, liposome nD20 was 1.590, and water was used as the buffer setting.

[0035] The average particle diameter of the charged particles B is 200 nm or less. By setting the average particle size of the charged particles B to 200 nm or less, the diffusion coefficient of the charged particles B in the solvent C becomes sufficiently small. Therefore, the charged particles B rapidly bind to the antigen in the solvent through the antigen-antibody reaction. As a result, the removal efficiency of the antigen is improved.

[0036] The average particle diameter of the charged particles B may be 100 nm or less, or 60 nm or less. The lower limit of the average particle size of the charged particles is not particularly limited, but may be 30 nm.

[0037] Charged particles B can be prepared by Bangham method or microfluidic method. Charged particles B used in the antigen recovery system in the solvent disclosed herein are dispersions in which charged particles are dispersed in liquid. Charged particles B before use can be freeze-dried particles or particles in an antifreeze state to which sugars such as sucrose are added.

[0038] <Solvent> Solvent C is blood from an organism. However, the solvent C is not necessarily limited to blood. As long as it is a liquid derived from a living body and contains a substance to be removed, a liquid other than blood may be used as the solvent C. When blood is used, the collected blood can be used directly or pre-treated blood can be used. Pre-treatment includes treatment required for the antigen recovery system in the solvent (for example, removing substances that reduce the efficiency of antigen recovery by the antigen recovery system A in the solvent).

[0039] In addition, when evaluating the performance of antigen retrieval systems in solvents, artificially prepared solvents are sometimes used in order to eliminate uncertainties. As an artificially produced solvent, there can be mentioned a PBS (physiological saline) solution containing Escherichia coli (antigen) at a certain concentration.

[0040] <Mixing section> The mixing section 4 is configured such that the charged particles B are put into the solvent C and the charged particles B and the solvent C are stirred in the mixing section 4 .

[0041] The mixing unit 4 may be a sealable container having a predetermined volume (eg, 10 to 50 mL). In this case, the charged particles B and the solvent C are mixed and stirred in the above-mentioned container. For example, a rotator having a rotating disk with adjustable rotation speed can be used for stirring. The container containing the charged particles B and the solvent C is fixed on the rotating disk, and the charged particles B and the solvent can be mixed and stirred by operating the rotator under prescribed conditions (temperature, time, rotation speed, etc.).

[0042] like Figure 1 As shown, flow paths 11, 12, 13, 14 and 15 may be respectively provided between the first tank 1, the mixing section 4, the pump 6, the recovery section 7 and the second tank 8, and the mixing section 4 may be assembled into a series of flows of the solvent C flowing from the first tank to the second tank. In this case, the mixing unit 4 includes a charged particle introduction unit 2 and a mixer 3 connected in series. The charged particle introduction unit 2 is provided between the first tank and the mixer 3 .

[0043] The charged particle introduction part 2 is a junction of three-way branches. The first of the three-way branches is a first flow path 11. Under the action of the pump 6, the solvent C before treatment stored in the first tank 1 flows in. The second of the three-way branches is a second flow path 12. Under the action of the pump 6, the solvent C in the charged particle introduction part 2 is transported from the charged particles B to the mixer 3.

[0044] The third of the three ports is a charged particle injection flow path from the syringe 5. By pushing the plunger of the syringe 5, the charged particles B in the syringe are injected into the charged particle injection unit 2 at a constant speed. The charging speed of the charged particles B (liposome dispersion) charged from the syringe 5 into the charged particle charging section 2 is 1 to 10 μm / min. In this case, the concentration of charged particles B in the charged particle input section 2 is 0.01 to 2.0 mg / ml. When the charged particle concentration is less than 0.01 mg / ml, the charged particles B (liposomes) input into the system are insufficient, and the recovery efficiency of the antigen is reduced. When the charged particle concentration exceeds 2.0 mg / ml, antibodies (free antibodies) that are not bound to the antigen are produced, and these antibodies saturate the adsorption carrier in the recovery section 7 (anionic adsorber). In this case, the antibody bound to the antigen cannot be fully recovered by the recovery unit 7, and the recovery efficiency of the antigen is reduced.

[0045] The mixer 3 is configured to stir the solvent C and the charged particles B before the antigen recovery process that have been combined in the charged particle input section 2. The mixer 3 may be a static mixer. By providing the mixer 3, the solvent C and the charged particles B before the antigen recovery process are effectively and fully mixed and stirred. When the solvent C and the charged particles B before the antigen recovery process are fully mixed and stirred before reaching the recovery unit, the mixer 3 may be omitted.

[0046] <Recycling Department> The recovery unit 7 is configured to recover the charged particles B mixed with the solvent C. The recovery unit 7 is an anionic adsorber including an adsorption carrier having a compound having an anionic functional group fixed on the surface thereof.

[0047] The recovery part 7 is in the shape of a tube of a predetermined length and includes an outer wall and an internal space. The length of the recovery portion 7 may be 10 to 300 mm, 30 to 200 mm, or 50 to 150 mm. The inner diameter of the recovery part 7 may be 10 to 100 mm, 15 to 70 mm, or 20 to 50 mm. The capacity of the internal space of the recovery part 7 may be 0.5 to 750 ml, 1 to 500 ml, or 5 to 100 ml.

[0048] The recovery part 7 includes an inlet for receiving the solvent C and the charged particles B mixed and stirred in the mixing part 4 , and an outlet for sending the processed solvent C after passing through the inside of the recovery part 7 to the outside of the recovery part.

[0049] The internal space of the recovery unit 7 is filled with an adsorption carrier having a compound having an anionic functional group fixed on the surface of the adsorption carrier. The adsorption carrier comprises a plurality of beads, and the particle size of the beads is 10 to 500 μm. The particle size may be 20 to 300 μm, or 40 to 200 μm.

[0050] The composition of the adsorption carrier is not particularly limited, and for example, cellulose, agarose, silica gel, activated carbon, etc. can be used.

[0051] The anionic functional group possessed by the compound fixed on the surface of the adsorption carrier may be a sulfate group, a carboxyl group, a phosphoric acid group, a sulfonic acid group or the like. The compound having an anionic functional group may be sodium dextran sulfate, sodium polyacrylate, sodium polyphosphate, sodium polystyrene sulfonate, sodium polyvinyl alcohol sulfate, or the like. Furthermore, any compound can be used as long as it is relatively easy to be fixed on the surface of the adsorption carrier and can impart a negative charge to the surface of the adsorption carrier.

[0052] The average zeta potential of the beads was below -20 mV. When the average zeta potential of the beads is −20 mV or less, the electrostatic interaction between the adsorption carrier and the charged particles B is enhanced, and the efficiency of antigen recovery in the recovery section 7 is improved.

[0053] The average zeta potential of the beads may be below -30 mV.

[0054] <Other components of the antigen retrieval system A in the solvent> Hereinafter, the configuration other than the charged particles B, the solvent C, the mixing unit 4 and the recovery unit 7 will be described.

[0055] The first tank 1 is a container for storing the solution C before antigen retrieval (antigen removal) treatment. The second tank 8 is a container for storing the solution C after the antigen retrieval (antigen removal) treatment. The pump 6 is a fluid pressure-feeding device, and transmits at least the pressure for pressure-feeding the solution C from the mixing unit 4 to the recovery unit 7 to the solution C in the system.

[0056] For example, a peristaltic pump suitable for aseptic liquid feeding can be used as the pump 6. As long as aseptic liquid feeding can be performed, a pump other than the peristaltic pump can also be used. The pump 6 is configured to be able to control the flow rate of the solvent C (in a fifth flow channel 15 described later) to be 100 ml / min or less. When the flow rate exceeds 100 ml / min, the force generated by the fluid becomes large, and the antigen adsorption efficiency in the recovery part 7 (anionic adsorber) decreases. The flow rate of solvent C may be 0.15 to 90 ml / min, or 1 to 50 ml / min.

[0057] The first tank is connected to the charged particle input unit 2 through a first flow path 11. The charged particle input unit is connected to the mixer 3 through a second flow path 12. The mixer 3 is connected to the pump 6 through a third flow path 13. The pump 6 is connected to the recovery unit 7 through a fourth flow path 14. The recovery unit 7 is connected to the second tank 8 through a fifth flow path 15.

[0058] (Antigen recovery method in solvent) Another embodiment of the present disclosure is a method for recovering antigens in a solvent. Figure 3 To illustrate the method of antigen recovery in this solvent. Figure 3 Detailed description of the invention is a flow chart showing the steps of a method for recovering an antigen in a solvent according to an embodiment of the present disclosure. In the description of the method for recovering an antigen in a solvent, the parts overlapping with those of the system for recovering an antigen in a solvent according to the above-mentioned embodiment are identical in content, and thus description thereof is omitted.

[0059] The method for recovering antigens in a solvent comprises: step S1, putting charged particles into a solvent and stirring; and step S2, recovering the charged particles mixed with the solvent. The charged particles contain 3 to 70 mol % of a cationic lipid, 1 to 20 mol % of a PEG-containing lipid, and 20 to 50 mol % of cholesterol. The surface of the above-mentioned charged particles is modified with antibodies. The average zeta potential of the above charged particles is -20 to +15 mV. The charged particles mixed with the solvent are recovered by electrostatic adsorption via an adsorption carrier having a compound having an anionic functional group fixed on the surface thereof.

[0060] <Mixing Step S1> In the mixing step S1 , the charged particles B are put into the solvent C and stirred. The charged particles B can be introduced into the solvent C by introducing the charged particles B into a container containing the solvent C. Alternatively, the charged particles B can be introduced into the solvent flow channels by merging the flow channels for introducing the charged particles into the flow channels.

[0061] In the former case, a mixing container (eg, a plastic microtube) having a predetermined capacity (eg, 0.1 ml to 2.0 ml) may be used as the container. The concentration of the charged particles B in the mixing container may be 0.01 to 5.0 mg / ml. After the charged particles B are added, the mixing container may be slowly turned upside down 5 to 10 times.

[0062] In the latter case, the concentration of the charged particles B in the charged particle input section 2 as the confluence portion can be set to 0.01 to 2.0 mg / ml.

[0063] After the charged particles B are added to the solvent C, the charged particles B and the solvent C are stirred. When the charged particles B are added to the mixing container containing the solvent C, stirring can be performed using a rotator. The rotator includes a rotating plate that rotates in the circumferential direction. The mixing container into which the charged particles are placed is fixed to a rotating plate by a clamp, and the rotating plate is rotated under predetermined conditions (temperature, speed, time, etc.), thereby stirring the contents of the mixing container.

[0064] When the flow path for introducing charged particles into the flow path of the solvent is merged and the charged particles B are introduced therefrom, a mixer 3 (for example, a static mixer) may be provided downstream of the charged particle introduction unit 2 . The mixer 3 may be omitted. In this case, the lengths of the flow paths 13 and 14 from the charged particle input unit 2 to the recovery unit 7 are set to be sufficient. The length may be, for example, 0.01 to 1 m. By setting the pump 6 , the time for the solvent C to flow from the charged particle input section 2 to the recovery section 7 can be set to 0.05 to 10 minutes.

[0065] <Recovery Step S2> In the recovery step S2, the charged particles mixed with the solvent are recovered. In the recovery step S2 , the charged particles B are adsorbed by electrostatic interaction between the positive charge caused by the cationic lipid contained in the charged particles B and the negative charge caused by the anionic functional group fixed on the surface of the adsorption carrier.

[0066] Among the charged particles mixed with the solvent, there are charged particles bound to the antigen contained in the solvent C and charged particles not bound to the antigen. In the recovery step S2, these two types of charged particles are recovered. Therefore, the recovery unit 7 has sufficient charged particle recovery capability for the charged particles B introduced so as not to be saturated with the charged particles. Example

[0067] The present invention will be described below based on more detailed examples, but the present invention is not limited to these examples.

[0068] (Example 1) (Evaluation was performed using PBS containing E. coli as a solution) First, E. coli was cultured and prepared. In a clean bench, add 10 ml of LB liquid culture medium to a 25 ml test tube, and add a small amount (a few μl) of E. coli inoculum solution. Then use an oscillator to culture E. coli at 37°C, 12 hours, and 140 rpm. Take 1 ml of the cultured E. coli solution and dispense it into a 1.5 ml tube. Centrifuge the tube at 4400 rpm for 5 minutes to precipitate the E. coli cells at the bottom of the tube, and remove the supernatant. Add 1 ml of PBS buffer to the tube and resuspend the E. coli cells precipitated at the bottom by pipetting.

[0069] Repeat the above steps from centrifugation to resuspension again. The concentration of E. coli in the second resuspended solution was adjusted to 8 × 10 8 CFU / ml. Then, the E. coli suspension was diluted 1000 times with PBS. The concentration of E. coli at this time was 8×10 5More specifically, 0.9 ml of PBS was added to 0.1 ml of the E. coli suspension to dilute it 10 times, and this operation was repeated 3 times.

[0070] Next, a liposome dispersion (aqueous solution containing charged particles) was prepared by the Bangham method. Specifically, the preparation was carried out by the following method. The constituent components of each liposome were stirred and dissolved in a mixed solution of chloroform and methanol at a specified ratio. The solvent was distilled off using an evaporator to produce a lipid film. The dispersion obtained from the lipid film was processed by an extruder through a hydrophilic polycarbonate membrane and granulated. Reduced anti-E. coli antibodies were added to the liposome dispersion. Stirred overnight under refrigeration. Free antibodies were removed by ultrafiltration in a solvent at room temperature. The solution was recovered. PBS was added to the previously prepared liposome dispersion (about 15 mg / ml) to adjust the liposome concentration to 1 mg / ml.

[0071] Then premix. 0.2 ml of the liposome dispersion (1 mg / ml) prepared in the above step was added to a 1.5 ml tube, and the above E. coli suspension (about 8 × 10 5 CFU / ml) 0.05ml. Then, fix the tube on the rotating plate of the rotator and stir for more than 30 minutes (20rpm). After stirring, use a pipette to take 0.2ml of the tube content and add it to PBS (5ml) in a container (volume: 5ml) prepared separately. Then, slowly turn it upside down about 10 times.

[0072] Then, a flow-through recovery test was performed. Assembled with Figure 1 A partial duplicate flow-through recovery test setup of the antigen recovery system in solvent is shown. Figure 1 The difference is that: the mixing section 4 is omitted; and the mixed solution (5 ml) of the above liposome suspension and Escherichia coli suspension is stored in the first tank.

[0073] The inlet side solution is sucked by a peristaltic pump (adsorption pump) and circulated in the anionic adsorber connected to the tube at a rate of 7.5 to 15 ml / h and discharged to the outlet side. After the circulation starts, samples are collected every about 1 minute from each elapsed time (e.g., 10 minutes / 20 minutes / 30 minutes).

[0074] Then, the live Escherichia coli concentration of the recovered samples was measured. 0.1 ml of the outlet side sample was collected at each elapsed time from the start of flow in the flow-through adsorption recovery test, and added to 0.9 ml of PBS to be diluted 10-fold. Spread 0.1 ml of the sample diluted 10 times evenly on the surface of the agar medium and culture it at 35°C for 22 to 24 hours. On the next day, the number of colonies formed was measured and the viable bacterial concentration was calculated according to the following formula: Three agar plates were used for each sample, and the viable bacterial concentration was calculated based on their average value. Live bacteria concentration (CFU / ml) = (colony count) × (1.0ml / 0.1ml) × (dilution ratio (10 times))

[0075] For the inlet side samples, the viable bacterial concentration was calculated using the same method. Based on the viable counts of Escherichia coli in the inlet and outlet samples, the adsorption removal rate was calculated according to the following formula. Adsorption removal rate (%) = 100 × {1-(live bacteria concentration of the outlet sample / live bacteria concentration of the inlet sample)} Specific adsorption removal rate (%) = 100 × {1-(live bacteria concentration at the outlet of E. coli antibody-modified liposomes (Example) / live bacteria concentration at the outlet of control antibody-modified liposomes (Comparative Example))} It should be noted that the compositions of the E. coli antibody-modified liposomes and the control antibody-modified liposomes were the same. The control antibody (isotype control) was used as a negative control for not recognizing E. coli as an antigen. Specifically, the same rabbit IgG isotype control antibody as the isotype control of the E. coli antibody was used.

[0076] Table 1 shows the solvents, antigens, and bacterial cell concentrations used in Examples A1 to A13 and Comparative Examples A1 to A12. Table 2 shows the modified antibodies (type and concentration), composition (type and content of each component), and properties (average zeta potential and average particle size) of the liposomes (charged particles) used in these Examples and Comparative Examples. Table 3 shows the premixing conditions, pump flow rate, solvent, and composition (types of surface functional groups and anionic compounds) and properties (average zeta potential and average particle size) of the anionic adsorber in these Examples and Comparative Examples. Table 4 shows the adsorption removal rates and specific adsorption removal rates evaluated in these Examples and Comparative Examples.

[0077] [Table 1]

[0078] [Table 2]

[0079] [Table 3]

[0080] [Table 4]

[0081] (Examples A1 to A13, Comparative Examples A1 to A12) In Examples A1 to A13 satisfying the conditions shown in Tables 1 to 3, by using appropriate liposomes (charged particles), a mixing section, and a recovery section, significantly higher adsorption removal rates than those of Comparative Examples A1 to A12 can be obtained. Comparative Examples A1 to A12 roughly correspond to Examples A3 to A11. In Comparative Examples A1 to A12, control antibodies were used as antibodies, and by comparing these corresponding Examples and Comparative Examples, the specificity of the recovery rates obtained in the Examples can be understood.

[0082] From the results of Examples A1 to A3, it can be seen that as the amount of liposomes added decreases, the adsorption removal rate decreases. In Comparative Examples A10 and A11, the content of cationic lipid in the liposome was too low, 0 mol% and 2 mol%, respectively, and the adsorption removal rate was extremely low, -5% to 11%, despite the use of E. coli antibodies.

[0083] In Comparative Example A12, hydroxyl groups were used as the surface functional groups of the anionic adsorber, and polyvinyl alcohol was used as the nonionic polymer (nonionic compound). The average zeta potential of the anionic adsorber in Comparative Example A12 was too low, at -10 mV, and the adsorption removal rate was extremely low, at -10% to 7%, despite the use of E. coli antibodies.

[0084] The effect of the in-solvent antigen recovery system of the present disclosure was evaluated using a PBS solution containing Escherichia coli as a solvent. The results showed that the antigen in the solvent can be efficiently recovered using liposomes with high biocompatibility.

[0085] (Example 2) (Evaluation was performed using bovine blood containing E. coli as a solution) The effect of the antigen recovery system in the solvent of the present disclosure was evaluated in substantially the same manner as in Example 1. The difference in Example 2 is that the PBS solution used as the solvent containing the antigen in Example 1 was replaced with bovine blood (pre-treated) or human blood (pre-treated). The description of other identical parts is omitted.

[0086] Cow blood and human blood contain substances that can lyse bacteria. When these bloods are directly used for the evaluation of this embodiment, not only will the number of viable Escherichia coli in the solvent be reduced by the antigen recovery system in the solvent, but also the reduction caused by the lysing effect, so they are not suitable as evaluation objects. Based on the above reasons, in this embodiment in which bovine blood or human blood is used as a solvent containing an antigen for the test, these liquids are pre-treated to avoid the above problems.

[0087] Specifically, using a flow-through device, bovine blood or human blood is passed through an anionic adsorber at a rate of 15 ml / h to partially remove components related to natural immune function contained in the bovine blood.

[0088] Then, Escherichia coli was cultured. In a clean bench, add 10 ml of LB liquid medium to a 25 ml test tube, and add a small amount (a few μl) of E. coli inoculum solution. Then, use an oscillator to culture the E. coli at 37°C, 12 hours, and 140 rpm. Take 1 ml of the cultured E. coli solution and dispense it into 1.5 ml tubes. The tube was centrifuged at 4400 rpm for 5 minutes to precipitate the E. coli cells at the bottom of the tube, and the supernatant was discarded. 1 ml of PBS buffer was added to the tube, and the E. coli cells separated at the bottom were resuspended by pipetting.

[0089] The concentration of E. coli in the second resuspended solution was adjusted to 8 × 10 8 CFU / ml. Then, the E. coli suspension was diluted 1000 times with PBS. The concentration of E. coli at this time was 8×10 5 More specifically, 0.9 ml of PBS was added to 0.1 ml of the E. coli suspension to dilute it 10 times, and this operation was repeated 3 times.

[0090] The same procedure as in Example 1 was used to prepare a liposome dispersion (aqueous solution containing charged particles). Pre-treated bovine blood was added to the previously prepared liposome dispersion (about 15 mg / ml) to adjust the liposome concentration to 0.5-2 mg / ml.

[0091] The premixing, flow-through recovery test and measurement of the live E. coli concentration of the recovered samples were performed in the same manner as in Example 1, except that pretreated bovine blood or pretreated human blood was used instead of PBS. Table 5 shows the solvents, antigens and bacterial concentrations used in Examples B1 to B13 and Comparative Examples B1 to B8. Table 6 shows the modified antibodies (type and concentration), composition (type and content of each component), and properties (average zeta potential and average particle size) of the liposomes (charged particles) used in these Examples and Comparative Examples. Table 7 shows the premixing conditions, pump flow rate, solvent and anionic adsorber compositions (types of surface functional groups and anionic compounds) and properties (average zeta potential and average particle size) in these Examples and Comparative Examples. Table 8 shows the adsorption removal rates and specific adsorption removal rates evaluated in these Examples and Comparative Examples.

[0092] [Table 5]

[0093] [Table 6]

[0094] [Table 7]

[0095] [Table 8] (Examples B1 to B11, Comparative Examples B1 to B8) In Examples B1 to B11 satisfying the conditions shown in Tables 5 to 7, higher adsorption removal rates than those of Comparative Examples B1 to B8 were obtained by using appropriate liposomes (charged particles), a mixing section, and a recovery section. In Comparative Examples B1 to B8, control antibodies were used as antibodies, and by comparing these corresponding Examples with Comparative Examples, the specificity of the recovery rates obtained in the Examples can be understood.

[0096] In Examples B8 and B9, it is estimated that the adsorption removal rate is reduced mainly because the content of the PEG-containing lipid in the liposome is lower than that in other Examples. In Examples B10 and B11, it is estimated that the adsorption removal rate is reduced mainly because the particle size of the liposome is larger than that of other Examples and the average zeta potential of the anion adsorber is lower than that of other Examples.

[0097] The effect of the antigen recovery system in the solvent according to the present disclosure was evaluated using bovine blood or human blood containing Escherichia coli as a solvent. The results showed that the antigen in the solvent can be recovered efficiently using liposomes with high biocompatibility.

[0098] (Example 3) (Evaluation of an integrated in-solvent antigen retrieval system) In Example 3, the antigen recovery system in a solvent according to the present disclosure in which the mixing unit 4 is integrated was evaluated. The difference between Example 3 and Example 1 is that the mixing unit 4 constructed as a separate container in Example 1 is integrated into the solvent circulation system of the antigen recovery system in the solvent. The description of other identical parts is omitted.

[0099] The culture and preparation of Escherichia coli were carried out in the same manner as in Example 1. A liposome dispersion (aqueous solution containing charged particles) was prepared in the same manner as in Example 1.

[0100] Assembly Figure 1 The device shown in the figure uses a peristaltic pump to circulate the prepared bacterial cell-containing solvent (PBS, blood) at a rate of about 10 ml / h and discharge it to the outlet side. Samples are collected 20 minutes, 30 minutes, and 40 minutes after the start of circulation. Liposomes are injected using a syringe 5 from 40 minutes after the start of circulation. Samples are collected 60 minutes after the start of circulation.

[0101] The viable count of Escherichia coli contained in the collected sample was measured in the same manner as in Example 1. In the evaluation of the antigen retrieval system in solvent, the post-injection adsorption removal rate defined by the following formula was also calculated. Adsorption removal rate after injection (%) = 100 × (live bacteria concentration at the outlet just before the injection of liposomes) / (live bacteria concentration at the outlet after the injection of liposomes)

[0102] Table 9 shows the modified antibodies (type and concentration), composition (type and content of each component), and properties (average zeta potential and average particle size) of the liposomes (charged particles) used in Examples C1 to C9 and Comparative Examples C1 to C2. The liposome concentration, injection rate, and concentration in the solvent after injection in these Examples and Comparative Examples are shown in Table 10. Table 10 also shows the pump flow rate, the composition (type of surface functional groups and anionic compounds) and properties (average zeta potential and average particle size) of the solvent and anionic adsorber. The adsorption removal rates after addition as evaluation of these Examples and Comparative Examples are shown in Table 11. When the adsorption removal rate after addition was 20% or more, it was determined that there was an adsorption enhancement effect after liposome addition, and was indicated by the symbol "○".

[0103] [Table 9]

[0104] [Table 10]

[0105] [Table 11]

[0106] (Examples C1 to C9 and Comparative Examples C1 to C2) In all Examples C1 to C9, the adsorption removal rate after input was increased by at least 20% compared with Comparative Examples C1 to C2. In Example C6, since the liposome input concentration, input speed, and concentration in the solvent after input are lower than those in other examples, it is estimated that the adsorption removal rate after input is lower than those in other examples. In Example C9, since the liposome introduction speed and the concentration in the solvent after introduction were too high compared to other examples, and the pump flow rate was also too fast compared to other examples, it is estimated that the adsorption removal rate after introduction was lower than that of other examples.

[0107] The system for recovering antigens in a solvent according to the present disclosure can efficiently recover antigens in a solvent using a medium with high biocompatibility even if the system is in a form in which the mixing unit 4 is integrated. Industrial Applicability

[0108] A predetermined component (antigen) in a solvent (eg, blood) can be effectively removed using a medium with high biocompatibility. Description of Reference Numerals

[0109] A Antigen Retrieval System in Solvent 1First Can 2 Charged particle input part 3. Mixer 4 Mixing section 5 Syringes 6 Pumps 7Recycling Department 8 Second Can B Charged particles (liposomes) 21 Cationic lipids 22 Neutral lipids 24 PEG-containing lipids 25 Antibodies C solvent (blood)

Claims

1. An antigen recovery system in a solvent, comprising charged particles, a mixing unit and a recovery unit, wherein the charged particles contain 3 to 70 mol% of a cationic lipid, 1 to 20 mol% of a PEG-containing lipid, 20 to 50 mol% of cholesterol and 0 to 65 mol% of a neutral lipid, The surface of the charged particle is modified with antibodies, The average zeta potential of the charged particles is -20 to +15 mV, The mixing section is configured such that the charged particles are put into a solvent and the charged particles and the solvent are stirred in the mixing section. The recovery unit is configured to recover the charged particles mixed with the solvent. The recovery unit is an anionic adsorber including an adsorption carrier having a compound having an anionic functional group fixed on the surface of the adsorption carrier.

2. The system for recovering antigens in solvent according to claim 1, wherein: The anionic functional group is a sulfate group, a carboxyl group or a sulfonyl group. The compound is dextran sulfate or polyacrylic acid or polystyrene sulfonic acid.

3. The system for recovering antigens in solvent according to claim 1, wherein: The charged particles contain 10 to 60 mol% of the cationic lipid, 6 to 14 mol% of the PEG-containing lipid, 20 to 50 mol% of the cholesterol and 0 to 30 mol% of the neutral lipid, The average zeta potential of the charged particles is -15 to +10 mV.

4. The system for recovering antigens in solvent according to claim 1, wherein: The charged particles contain 20 to 60 mol% of the cationic lipid, 8 to 14 mol% of the PEG-containing lipid, 20 to 50 mol% of the cholesterol, and 0 to 25 mol% of the neutral lipid, The average zeta potential of the charged particles is -10 to +5 mV.

5. The system for recovering antigens in solvent according to claim 1, wherein: The average particle size of the charged particles is less than 200 nm.

6. The system for recovering antigens in solvent according to claim 1, wherein: The average particle size of the charged particles is 100 nm or less.

7. The system for recovering antigens in solvent according to claim 1, wherein: The average zeta potential of the adsorption carrier is below -20 mV.

8. The system for recovering antigens in solvent according to claim 1, wherein: The average zeta potential of the adsorption carrier is below -30 mV.

9. The system for recovering antigens in solvent according to claim 1, wherein: The concentration of the charged particles in the mixing section is 0.01 to 2.0 mg / ml.

10. The system for recovering antigens in solvent according to claim 1, wherein: The mixing part is connected to the recovery part through a first flow path, The mixing section is also connected to the second flow path. The recovery unit is also connected to the third flow path. The antigen recovery system in a solvent includes a pump configured to control a flow rate of the solvent, and the pump is configured to adjust the flow rate of the solvent in the third flow path to 100 ml / min or less.

11. A method for recovering an antigen in a solvent, comprising: The step of adding charged particles into a solvent and stirring; and a step of recovering the charged particles mixed with the solvent, The charged particles contain 3 to 70 mol% of cationic lipids, 1 to 20 mol% of PEG-containing lipids, 20 to 50 mol% of cholesterol and 0 to 65 mol% of neutral lipids. The surface of the charged particle is modified with antibodies, The average zeta potential of the charged particles is -20 to +15 mV. The charged particles mixed with the solvent are recovered by electrostatic adsorption via an adsorption carrier having a compound having an anionic functional group fixed on the surface thereof.

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