JP7342448B2 - Particle detection method - Google Patents

Description

The present invention relates to a particle detection method.

The development of pharmaceuticals using nanoparticles such as polymeric micelles and liposomes has been progressing for many years, and some liposomes in particular are actually widely used as pharmaceuticals around the world. Pharmaceutical treatments using these nanoparticles are called drug delivery systems (hereinafter referred to as DDS), and are attracting attention as they enable treatments with reduced side effects by efficiently delivering drugs to the affected area through enhanced permeability and retention effects. Bathing. On the other hand, since it is a drug that uses nanoparticles, quality control that takes into account particle aggregation, association, and fusion is required compared to general low-molecule drugs.

Particle size measurement, which is one of the quality control items for nanoparticle medicines, is generally performed by dynamic light scattering (hereinafter referred to as DLS) or laser diffraction scattering, which detects scattered light obtained from a group of particles and determines the particle size distribution. Since this method calculates , it is not suitable for detecting minority particle groups.

For detection of minority particle groups, techniques that measure each particle one by one are thought to be more accurate; for example, nanotracking analysis, which observes the Brownian motion of each particle, and the mass detection of individual particles. A resonant mass measurement method for measuring , and a Coulter method (electronic sensing zone method; hereinafter referred to as ESZ) using electrical detection are known (for example, see Non-Patent Document 1). In these methods, the information (signal) obtained from each detection means corresponds to each particle on a one-to-one basis, so it is possible to evaluate each particle individually, and it is possible to evaluate the proportion contained numerically. Although accurate measurement is possible even with a small number of particles, there is a problem that the measurable particle size range (dynamic range) is relatively narrow.

R. W. De Blois. et al, The Review of Scientific Instruments, Volume 41, Number 7, pp909-916 (1970)

An object of the present invention is to provide a particle detection method that can individually and continuously detect particles with a wide distribution contained in nanoparticle medicines.

The method for detecting particles contained in nanoparticle medicines according to the present invention involves separating particles in a direction perpendicular to a fluid flow according to their particle diameter, and dividing the separated particles into two or more flow channels. The method is characterized in that the particles are detected by a particle detection section installed in the flow path.

Further, in one aspect of the method for detecting particles contained in a nanoparticle drug according to the present invention, the particles are detected with an electric detector including electrodes placed across an aperture installed in the flow path. .

Furthermore, in one aspect of the method for detecting particles contained in nanoparticle pharmaceuticals according to the present invention, the particle diameter range that can be detected by the electric detector varies depending on the installed flow path.

Furthermore, in one embodiment of the method for detecting particles contained in nanoparticle pharmaceuticals according to the present invention, part of the particle size ranges that can be detected by the electric detector overlap.

Furthermore, in one aspect of the method for detecting particles contained in nanoparticle pharmaceuticals according to the present invention, at least one parameter among the number of channels, the shape, width, height, and length of the branched portion is adjusted. The particles are separated into two or more channels by creating a channel structure that prevents particles of a certain size or more from being mixed in.

Furthermore, in one embodiment of the method for detecting particles contained in nanoparticle pharmaceuticals according to the present invention, particles are separated using the principle of pinched flow fractionation.

Furthermore, the present invention detects at least one of particles, aggregates, aggregates, and fusions in nanoparticle pharmaceuticals by the method described above, and detects particle size distribution, average particle size, mode size, intermediate particle size, and The method includes a method of measuring at least one parameter among the volume ratio, concentration, number, cumulative concentration, and cumulative number of the sample, or a change in the parameter over time.

The present invention makes it possible to continuously detect particles with a wide range of particle size distribution, minority particles, and particle groups in nanoparticle medicines with high precision. This makes it possible to measure large particle samples.

FIGS. 1A and 1B are diagrams showing an embodiment of the present invention, in which hydrodynamic filtration (HDF) is applied to a particle separation channel 110. FIG. 1(a) is a top view of the microchip 10, and FIG. 1(b) is an enlarged view of the region 190 in FIG. 1(a). FIG. 2(a) is a diagram showing an embodiment of the present invention, and shows a mode in which two particle detection units 103 are arranged in series. FIG. 2(b) is a diagram schematically showing how the measured current value changes over time when one particle passes through the two particle detection units 103 continuously, and the upper graph is The graph on the lower side indicates the measurement by the particle detection unit 103 on the downstream side. Here, for convenience, the positions of the vertical axes of the graphs of the two particle detection units 103 are shifted, but in reality, the current values serving as the baseline when no particles pass are almost the same. FIG. 3(a) is a diagram showing an embodiment of the present invention, in which two apertures are arranged in series, and unlike FIG. 2, it shows a mode in which only one pair of electrodes is used. FIG. 3(b) is a diagram schematically showing how the measured current value changes over time when particles successively pass through two apertures. FIG. 4(a) is a diagram showing an embodiment of the present invention, and shows a mode in which a plurality of particle detection sections 103 are installed in parallel for one particle collection channel 102. FIG. 4(b) shows an equivalent circuit of FIG. 4(a). FIG. 5(a) is a diagram showing one embodiment of the present invention, and shows a mode in which a plurality of particle detection units 103 are installed in parallel to one particle collection channel 102, and a plurality of particle A mode is shown in which the detection unit 103 reduces electrical interference between a plurality of particle detection units 103 by applying a voltage from an electrode on the fluidically downstream side. FIG. 5(b) shows an equivalent circuit of FIG. 5(a), and shows that the plurality of particle recovery channels 102 are independent. FIG. 6 is a diagram showing an embodiment of the present invention, in which a plurality of apertures are installed in parallel for one particle collection channel 102, and shows an aspect that can be used as a lower cost device. FIG. 7 shows a mode for manufacturing the particle detection unit 103 at a lower cost using the relay flow path 60 and the electrode insertion port 59. FIGS. 8(a) to 8(c) are diagrams showing one embodiment of the present invention, and show an aspect in which HDF is applied in the particle separation channel 110. 8(a) shows a case with two particle collection channels 102, FIG. 8(b) shows a case with three particle recovery channels 102, and FIG. 8(c) shows a case with two particle recovery channels 102. is three and the particle separation channel 110 has a branch channel 105. FIG. 9(a) is a diagram showing the flow of fluid at the branch portion 110A in FIG. 8(b), and FIG. 9(b) is an enlarged view of the straight flow path portion in FIG. 9(a). FIG. 10(a) is a diagram showing the flow of particles in a straight channel under laminar flow conditions, and FIG. 10(b) shows the trajectory of the particle flow in the particle diffusion channel 110B. FIGS. 11(a) to 11(c) are diagrams showing one embodiment of the present invention, and show an aspect in which PFF is applied in the particle separation channel 110. 11(a) shows a case with two particle recovery channels 102, FIG. 11(b) shows a case with three particle recovery channels 102, and FIG. 11(c) shows a case with two particle recovery channels 102. An enlarged view of region 21 in (b) is shown. FIG. 12(a) is a diagram showing an embodiment of the present invention, and shows an aspect in which an asymmetric PFF is applied in the particle separation channel 110. FIG. 12(b) shows an enlarged view of region 21 in FIG. 12(a). FIG. 13(a) is a diagram showing an embodiment of the present invention, in which an asymmetric PFF is applied in the particle separation channel 110 and the wall surface on the narrow channel wall surface 16b side of the expanding channel 17 gradually expands. The mode is shown below. FIG. 13(b) shows an enlarged view of region 21 in FIG. 13(a). FIG. 14 is a diagram showing an embodiment of the present invention, in which a particle detection unit 103 is configured with two apertures, the downstream side of each aperture is connected to an outlet, and each outlet also serves as an electrode insertion port 59. This shows how it works. FIG. 15(a) shows a schematic diagram of the microchip 10 used in Example 1. FIG. 15(b) shows an enlarged view of region 21 in FIG. 15(a). FIG. 16 shows the particle size distribution of the treated sample created from the measurement results in Example 1. FIG. 17 shows the particle size distribution of the untreated sample created from the measurement results in Example 1. FIG. 18 shows the particle size distribution of the treated sample created from the measurement results in Comparative Example 1. FIG. 19 shows the particle size distribution of the untreated sample created from the measurement results in Comparative Example 1. FIG. 20 shows the particle size distribution of the heat-treated antibody drug for verifying changes over time, created from the measurement results in Example 2, with FIG. 20(a) showing 0 minutes after treatment, and FIG. 20(b) showing treatment. 10 minutes after treatment, and FIG. 20(c) shows 120 minutes after treatment. FIG. 21 shows the particle size distribution of the heat-treated antibody drug for verifying changes over time, created from the measurement results in Comparative Example 2, with FIG. 21(a) showing 0 minutes after treatment, and FIG. 21(b) showing treatment. 10 minutes after treatment, and FIG. 21(c) shows 120 minutes after treatment. Figure 22 shows the particle size distribution of heat-treated antibody pharmaceuticals for comparison of antibody aggregate amounts created from the measurement results in Example 3, where Figure 22(a) is antibody pharmaceutical A and Figure 22(b) is Antibody drug B, FIG. 22(c) shows the results for antibody drug C. Figure 23 shows the particle size distribution of heat-treated antibody pharmaceuticals for comparing the amount of antibody aggregates created from the measurement results in Comparative Example 3, where Figure 23(a) is antibody pharmaceutical A and Figure 23(b) is Antibody drug B, FIG. 23(c) shows the results for antibody drug C. FIG. 24 shows the results of evaluating the thermal stability of the antibody drug using a differential scanning calorimeter in Reference Example 1. FIG. 25(a) shows the particle size distribution of the untreated liposome preparation created from the measurement results in Example 4, and FIG. 25(b) shows the particle size distribution of the stored liposome preparation in Example 4. . FIG. 26(a) shows the particle size distribution of the untreated liposome preparation created from the measurement results in Comparative Example 4, and FIG. 26(b) shows the particle size distribution of the stored liposome preparation in Comparative Example 4. .

The present invention is a method for detecting particles contained in nanoparticle medicines, in which particles are separated in a direction perpendicular to a fluid flow according to their particle diameters, and the separated particles are passed through two or more flow channels. The flow path is divided into two parts, and the particles are detected by a particle detection unit installed in the flow path. An embodiment of the present invention will be described below.

A nanoparticle drug (hereinafter sometimes referred to as a sample) refers to a fluid containing a substance at least partially having a nano-order width and/or height, such as a polymer, micelle, liposome, etc. Refers to fluids containing antibodies, antigens, nucleic acids, peptides, proteins, viruses, virus-like particles, cells, or complexes formed with two or more of the above substances, and which are ultimately intended to be used as medicines, or Refers to a fluid containing the above-mentioned substances that can be candidates for pharmaceutical products. Nanoparticle medicines may contain aggregates, associations, fusions, etc. of the aforementioned particles, and the particle size is distributed in the range of 1 nm to 100 μm.

Furthermore, "separating in the vertical direction" does not necessarily have to be strictly perpendicular to the flow of the fluid, but it is sufficient that the particles are separated in a direction approximately perpendicular to the flow of the fluid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described in detail with reference to FIG. 1, which is a schematic diagram showing an example of the device configuration of a particle detection device. The cross section of the channel in the microchip 10 is preferably rectangular from the viewpoint of ease of manufacturing the channel structure, but it may also be a circular, oval, or polygonal cross section, or it may be partially rectangular. It may be of any other shape. Further, although it is preferable that the channel height is uniform for ease of fabrication, the depth may be partially different.

The sample is introduced from the inlet 14 and sent to the downstream side of the flow path by the liquid sending section, and is passed through the particle introduction channel 101, the particle separation channel 110, the particle recovery channel 102a or 102b, and the corresponding particle detection section 103a or 102b, respectively. 103b and flows out to outlet 104a or 104b. In the particle separation channel 110, particles are separated in a direction perpendicular to the fluid flow according to the particle diameter, and the separated particles flow to the particle recovery channel 102a or 102b. Electrical detection is performed in the particle detection units 103a and 103b that have arrived via the particle recovery channel 102a or 102b. At this time, the insides of the particle detection units 103a and 103b are filled with a solution containing an electrolyte, and are connected to an electric measuring device 56 and a power source 57 via conductive wires 55 connected to electrodes 54a and 54b, respectively. When detecting particles, a current of an arbitrary value is flowing from the power source 57, and a closed circuit is formed through the aperture 53 (see FIG. 2). Furthermore, the electric measuring device 56 is connected to an analysis section 61, and the detection signal obtained from the electric measuring device 56 is calculated by the analysis section 61 to create a particle size distribution.

The inlet 14 may have any structure as long as it can hold a sample, and preferably has a concave structure. Further, as the material, metal, glass, or ceramics, which have a small amount of eluate, may be used, but it is preferable to use a polymeric material in order to manufacture it at low cost. Particle introduction channel 101 is formed between inlet 14 and particle separation channel 110. Although the particle introduction channel 101 is arranged to assist the separation of particles in the particle separation channel 110, it may be omitted in order to downsize the microchip 10.

The liquid feeding section may use a method of feeding liquid using a pressure gradient such as a syringe pump, peristaltic pump, or pressure pump, or an electroosmotic flow pump may be used to suppress uneven velocity distribution in the cross section of the flow path of the microchip 10. may also be used. In this case, the piping connected from the pump is directly connected to the inlet 14 to apply pressure to the sample held in the inlet 14 to feed the sample. Alternatively, a pump may be connected to the outlet via piping, and the fluid in the flow path of the microchip 10 may be sucked by applying negative pressure to send the fluid. Furthermore, by making the liquid level in the inlet 14 higher than the liquid level in the outlet 104a or the outlet 104b, liquid may be fed based on the liquid level difference, and in this case, a liquid feeding part is not required. In order to perform more quantitative measurements, it is preferable to allow particles to pass through using a pressure gradient, and it is most preferable to use a pressure pump that causes less pulsation.

The flow rate of the liquid feeding section is preferably set to an arbitrary value depending on the cross-sectional area of the flow path and the cross-sectional area of the aperture, and is preferably set between 0.1 μL/hour and 1 mL/hour, as an example.

Two or more particle recovery channels 102 are provided in order to expand the measurable particle size range (dynamic range). Here, the particle detection unit 103 is provided inside or downstream of the particle recovery channel 102 and is used to detect particles flowing into the particle recovery channel 102. At least one particle detection section 103 must be provided for one particle collection channel 102, and two or more particle detection sections 103 may be provided (FIGS. 2 to 6). Furthermore, a flow path for collecting particles outside the measurable particle size range can be further provided. In an embodiment in which two or more particle detection units 103 are provided for one particle collection channel 102, the two or more particle detection units 103 may be arranged in series (FIGS. 2 and 3). For example, when two particle detection units 103 are arranged in series, the passage speed of particles in the flow path can be calculated from the interval between signals generated from the same particles that have passed through the two particle detection units 103. Since the particle passage velocity is generally proportional to the flow rate in the recovery channel 102, it is possible to estimate the flow rate of the fluid in the recovery channel. At this time, as shown in FIG. 2, the voltage applied to the electrodes is preferably applied from an electrode on the upstream side of the aperture 53' on the upstream side and an electrode on the downstream side of the aperture 53'' on the downstream side. When a voltage is applied from the downstream electrode 54b' of the upstream aperture 53' or the upstream electrode 54a'' of the downstream aperture 53'', a voltage drop occurs between the two electrodes, and one aperture Since a change in the current value of one aperture affects the current value of the other aperture, it is necessary to take measurements while taking these influences into account. Therefore, when using the particle detection unit 103 including two or more apertures, as shown in FIG. The measurement may be performed using only the electrode 54b''. As shown in FIG. 7, electrode insertion ports 59 are provided upstream and downstream of the particle detection channel, respectively, and are fluidly and electrically connected to the aperture via the relay channel 60, into which the electrodes 54 are inserted. Particle detection may be performed by ESZ by immersing the sample.

Further, in an embodiment in which two or more particle detection units 103 are provided for one particle collection channel 102, the two or more particle detection units 103 may be arranged in parallel (FIGS. 4 to 6). This aspect provides an excellent effect in that the amount of processing increases in proportion to the number of parallel particle detection units 103. At this time, electrodes 54 for electrical detection are arranged to sandwich each aperture, and measurement is possible if the number is twice the number of particle detection units 103. On the other hand, as shown in FIG. 6, it is also possible to perform measurement by arranging one electrode on the upstream side of each aperture and the same number of electrodes as the number of particle detection units 103 on the downstream side. This embodiment is preferable from the viewpoint of cost reduction through reduction. Here, if the particle detection units 103 are parallelized and a voltage is applied to each aperture at the same time, if voltage is simultaneously applied to a plurality of electrode pairs arranged to sandwich each aperture, as shown in FIG. 4(a), , the equivalent circuit becomes complex, and changes in the current value of one aperture affect the current values of other apertures, so it is necessary to take these effects into consideration. On the other hand, if you want to apply voltage to each aperture individually and detect each aperture individually, this can be done by leaving the aperture that is not being measured in an open circuit state without connecting it to ground or the chassis. There will be no impact on At this time, to create an open circuit state, a switching circuit may be used between the electrode 54 and the power source 57, or a relay system or a photoMOS system may be used to create an open circuit state. It is preferable for the particle detection unit 103 to construct a measurement system in which electromagnetic noise does not affect the measurement as much as possible.In this respect, it is preferable to construct a measurement system in which electromagnetic noise is not affected as much as possible during measurement. method is preferable. However, as shown in FIG. 5, when a voltage is applied from the electrode on the downstream side of the aperture toward the upstream side, a change in the current value of one aperture does not affect the current value of other apertures, so this embodiment is not applicable. May be used.

Particle detection section 103 includes an aperture 53 and an electric detector. Aperture 53 refers to a hole formed within the flow path that is smaller than the flow path diameter, and is defined by particle detection flow path 62 and aperture forming structure 52 . The cross-sectional shape of the aperture may take various shapes depending on the manufacturing process, and when processed by etching or laser irradiation, it takes a circular or elliptical shape, and when processed by photolithography and soft lithography, it takes the shape of a polymer such as polydimethylsiloxane (hereinafter referred to as PDMS). In the case of molding with material, it will be a rectangle. The cross-sectional area of the aperture only needs to be larger than the particles to be measured, but the particle diameter range that can be measured with ESZ is generally said to be 2 to 60% of the aperture cross-sectional area, so It is necessary to design according to the size of the Further, as shown in FIGS. 2, 3, and 14, the particle detection unit 103 may be configured with two apertures. Further, as shown in FIGS. 4 to 6, a plurality of apertures are formed by providing a plurality of particle detection sections, and the downstream end of each aperture is connected to an outlet, and each outlet also functions as an electrode insertion port 59. It is also possible to adopt a configuration in which this is done. In this case, if the volumes of the apertures are approximately the same, the signals obtained from each aperture will be approximately the same. That is, it is possible to detect all the particles that have flowed into the recovery channel upstream of each aperture, and this can be said to be a preferable embodiment from the viewpoint of quantitatively measuring the concentration. The sensitivity of particle detection by ESZ decreases in proportion to the flow path resistance between the aperture and the electrode, so when calculating the particle diameter from the obtained signal, it is necessary to take this signal decrease into account (formula (See (1)). At this time, L is the length of the flow path forming the aperture, de is the equivalent diameter of the aperture, L' is the length of the relay flow path 60, and de' is the equivalent diameter of the relay flow path 60. Further, it is preferable to take into account the signal reduction according to the formula (1) as appropriate depending on the channel structure, and the formula does not necessarily have to completely match the formula (1).

The electric detector of the particle detection unit 103 mainly includes an electrode 54 , an electric measuring device 56 connected to the electrode 54 via a conductive wire 55 , and a power source 57 . The two electrodes 54 are arranged with the aperture 53 in between. The electrical measuring device 56 may be any device that detects electrical characteristics, and examples thereof include a current measuring device, a voltage measuring device, a resistance measuring device, and a charge amount measuring device.It is preferable to use a current measuring device in measuring ESZ. Most preferred. Furthermore, it is preferable to use an IV amplifier to increase the gain after current-voltage conversion and to detect minute changes in current value in order to detect even minuter particles. In addition, in order to detect all the particles that have passed through the aperture, the sampling time interval of the electric measuring device 56 is preferably sufficiently shorter than the time required for the particles to pass through the aperture, and is 10,000 times per second. It is preferable to sample at least 20,000 times per second, and it is more preferable to sample at least 20,000 times per second.

The analysis unit 61 can include a calculation device for calculating measurement results and a recording medium for recording measurement results or calculation results derived therefrom. Alternatively, these computing devices and recording media may be integrated with the electrical measuring device 56 or may be external devices connectable to the electrical measuring device 56. The data recorded on the recording medium includes the sampled current value, the change in current value that occurs when the particles pass, and the particle diameter, number of particles, particle concentration, and detection time or measurement calculated from the change in current value. Contains the elapsed time since the start.

When hydrodynamic filtration (HDF) is used in the separation method used in the particle separation channel 110, the upstream end of the particle separation channel 110 is connected to the particle introduction channel 101, and the fluid flows out. It is sufficient if it is connected to the particle recovery channel 102 via the branch section 110A at the downstream end (FIG. 8). At this time, the branch part 110A needs to be fluidly connected to at least two or more particle recovery channels 102 in order to separate the particles, and the downstream fluid dynamic resistance including the particle recovery channels is reduced. It is necessary to take this into consideration when setting the cross-sectional area and volume of each flow path and aperture. For example, when three recovery channels 102 are provided as shown in FIG. 8(b) and FIG. 9, if the flow rates flowing to each recovery channel are Qa, Qb, and Qc, the ratio of each flow rate is the width w of the channel, It is calculated from the height h and the length L, and specifically, the flow rate in the straight flow path is calculated from the Hagen-Poiseuille equation according to equation (2).

Furthermore, as shown in the enlarged diagram 9(b), the velocity distribution in the flow path of the microchip under laminar flow conditions becomes a parabola, which is generally expressed as u(r) in equation (3) (at this time, w0 is the radius of the circular tube, r is the distance from the center of the circular tube, μ is the viscosity, L is the length of the circular tube, and ΔP is the pressure loss.)

The ratio of the areas Sa, Sb, and Sc within the parabola separated by arbitrary distances w1 and w2 from the channel wall surface is equal to the ratio of the flow rates Qa, Qb, and Qc flowing into each recovery channel. At this time, among the particles existing in the channel, particles whose center or center of gravity is closer to the channel wall surface than w1 flow into the recovery channel 102a, and are closer to the channel wall surface than w2. Particles existing between w1 and w2 flow into the recovery channel 102b, and particles existing between w1 and w2 flow into the recovery channel 102c. Therefore, the radii of the largest particles flowing into the recovery channels 102a and 102b are w1 and w2, respectively, so if the cross-sectional shape of the aperture is circular, the radius of the aperture must be greater than w1 or w2, and the radius of the aperture must be greater than or equal to w1 or w2. When the cross-sectional shape is approximately circular or elliptical, the minimum radius passing through the center or center of gravity of the approximately circular or elliptical shape must be greater than or equal to w1 or w2. Further, when the cross-sectional shape of the aperture is rectangular, each of the two sets of opposing sides needs to be at least twice as long as w1 or w2. In addition, if the aperture has a polygonal cross-sectional shape, the radius of its inscribed circle must be greater than or equal to w1 or w2.

Here, a mode may be adopted in which a plurality of branch portions 110A (not shown in FIG. 8) are provided at a location other than the end of the particle separation channel 110, and one or more branch channels 105 are provided (FIG. 8(c)). . The branch channel 105 may be provided only on one channel wall side, or may be provided on both sides of the channel wall surface. Further, one or more branch channels 105 may merge simultaneously in the downstream recovery channel 102, or may gradually merge.

Furthermore, a particle diffusion channel 110B may be formed fluidly upstream of the branch portion 110A (not shown in FIG. 10) of the particle separation channel 110 (FIG. 10(b)). The fluid upstream side of the particle diffusion channel 110B is connected to the particle introduction channel 101, and the fluid downstream side is connected to the branch section 110A. Furthermore, in the case of an embodiment in which the particle introduction channel 101 is not used, the fluid upstream side of the particle diffusion channel 110B is directly connected to the inlet.

Preferably, the particle diffusion channel 110B has a structure in which the width or height, or both, of the channel increases from the fluid upstream side to the downstream side. The preferred embodiment of this differs depending on whether a plurality of particle collection channels exist in the width direction of the particle diffusion channel 110B or a plurality of particle recovery channels exist in the height direction. When using a chip channel forming technique, a structure in which the width of the channel increases is preferable in terms of ease of fabrication.

In this particle diffusion channel 110B, by utilizing the Brownian motion of particles per unit time, that is, the fact that the diffusion distance is inversely proportional to the square root of the particle diameter, when the particles flow through the particle diffusion channel 110B, the particles Since particles diffuse in the direction in which the channel expands according to their diameter, the probability of existence of particles with small particle diameters increases near the enlarged channel wall surface, resulting in a concentration effect. Here, the angle θa at which channel wall surface a and particle diffusion channel wall surface a connect, and the angle θb at which channel wall surface b and particle diffusion channel wall surface b connect, in FIG. 10(b), expand the channel width. It must be less than 180° at the point. In addition, under high flow conditions where the flow velocity in the channel surrounded by channel wall surface a and channel wall surface B is 1 m/sec or more, there is a concern that vortex flow may occur in the enlarged part, so It is preferable to send the liquid under relatively low flow conditions such as less than 90 degrees, or to make the angles θa and θb larger than 90°. On the other hand, when particles flow into the particle diffusion channel 110B, the particles exert an inertial force in the direction in which the particles flow (hydrodynamic downstream direction, in which the branch section 110A exists) according to their weight. receive. In other words, when the density of particles is the same, particles with larger diameters receive inertial force in a direction closer to the center of the flow channel, so the proportion of particles with smaller diameters increases on the channel wall surface, resulting in further separation in HDF. Contribute to improving abilities. Further, the angles θa and θb may be asymmetrical, and one wall surface may be connected in a substantially straight line, and only the other opposing wall surface may be enlarged and connected at an angle of less than 180°.

When HDF is used in the separation method used in the particle separation channel 110, the particle size ranges that can be detected by the particle detection units 103a and 103b in FIG. It is preferable to set the measurement range so that different particle size ranges can be measured, and it is even more effective if some of the ranges overlap with each other in that it is possible to detect particles that could not be separated and obtain more robust results. preferable. As shown in FIG. 8(a), when there are two particle collection channels 102, the particle size range that can be detected by the particle detection units 103a and 103b is as follows: 103b may be set in a small particle size range, and vice versa without any problem. However, based on HDF theory, it is necessary to set the aperture so that it is not blocked by particles.

When converting the concentration from the number of particle counts, the flow rate flowing into each particle collection channel 102 is calculated using the set flow rate of the liquid feeding section and equation (2), so the number of particle counts per measurement time can be calculated using formula (2). By dividing by this, the particle concentration is calculated.

Next, FIG. 11 shows an example of a configuration in which a flow path using the principle of pinched flow fractionation (PFF) is used in the particle separation flow path 110. When using a channel using the principle of PFF, the particle separation channel 110 includes a branch channel 18a, a branch channel 18b, a narrow channel 16, and an enlarged channel 17. Inlets 14a and 14b holding particle-containing fluid 100P and particle-free fluid 100N are fluidly connected to branch channel 18a and branch channel 18b, respectively. The branch flow path 18a and the branch flow path 18b merge downstream thereof and are fluidly connected to the upstream side of the narrowed flow path 16. Thereafter, the particles are separated in an expanded channel 17 connected to the downstream side of the constricted channel 16 based on the principle of PFF. The channel width of the enlarged channel 17 is constant at a predetermined position, and particles are collected in recovery channels 102a and 102b that are fluidly connected downstream of the enlarged channel 17, and are Particles are detected by the detection units 103a and 103b. Here, any number of recovery channels may be installed as long as they are two or more, but from the viewpoint of reducing the size and cost of the channel, it is preferable to use five or less, and most preferably three or less.

Furthermore, the enlarged flow path 17 may be formed based on the principle of an asymmetric PFF. In this case, as shown in FIGS. 12 and 13, a drain channel 22 may be installed in the enlarged channel 17, and this embodiment is more preferable in terms of improving particle separation performance. The drain channel 22 is connected to the outlet 23, and fluid is discharged or collected. It is preferable to design so that 50% or more of the fluid flows into the drain channel, and more preferably to design so that 70% or more of the fluid flows into the drain channel.

Furthermore, the structure in which the narrowed channel 16 and the enlarged channel 17 are connected may be configured such that the channel width gradually increases as shown in FIG. 11(c) or FIG. 13(b). When the flow path width is gradually expanded, the wall surface 17b of the expanded flow path may be expanded to draw a straight line to form a slope 40, or may be expanded to draw a curve. When the channel width of the enlarged channel 17 expands stepwise or linearly, between the wall surfaces 16a, 16b of the narrowed channel and the wall surfaces 17a, 17b of the enlarged channel 17 following the wall surface. An enlarged flow path can be defined by the angles 24a and 24b. For example, the angles 24a and 24b in the case of stepwise expansion are 90°. When the wall surface of the enlarged channel 17 gradually expands linearly, the angles 24a and 24b are expressed as 90° to 180°. The angles 24a and 24b of the walls of the enlarged region can independently take any angle between 90° and 180°, and can take, for example, 120°, 135°, 150°, and 180°. However, even if the angle 24b of the enlarged channel wall surface 17b with respect to the narrowed channel wall surface 16b is 90 degrees or less, this is a partial structure and the structure gradually expands. If so, there is no problem because most of the fluid gradually flows toward the outlet 23.

At least two of the plurality of particle recovery channels 102 connected downstream of the enlarged channel 17 need to be provided, and it is preferable to arbitrarily increase the number depending on the particle diameter range to be measured. Each particle recovery flow path 102 has at least one particle detection section 103, and may have a plurality of particle detection sections.

The enlarged channel 17 may also have the function of the particle diffusion channel 110B described in the HDF section, thereby providing a function of further promoting the particle separation ability, but in this case, the enlarged channel 17 The length must be long enough to allow particles to diffuse, and must be at least 1 μm or more. The channel length of the enlarged channel 17 is determined to be an arbitrary value depending on the channel width to be expanded and the angles 24a and 24b.

Alternatively, a plurality of particle recovery channels 102 may be provided directly downstream of the narrowed channel 16 without using the expanded channel.

When a flow path using the principle of PFF is used for the particle separation flow path 110, the diameter of the particles that can exist increases from the narrowed flow path wall surface 16a side to the 16b side. In case a), it is preferable to set the cross-sectional area of the aperture so that the particle detection unit 103a detects small-diameter particles and the particle detection unit 103b detects large-diameter particles. In the case of FIG. 11(c), the cross-sectional area of the aperture is set such that the particle detection unit 103a detects small-diameter particles, the particle detection unit 103c detects medium-diameter particles, and the particle detection unit 103b detects large-diameter particles. It is preferable to set In other words, it is preferable to set the cross-sectional area of the aperture so that the closer the particle detection section 103 is to the narrowed channel wall surface 16a, the smaller particles can be detected. For example, when trying to detect particles with a diameter of 0.1 to 2.0 μm, in the case of FIGS. 11(c), 12, and 13, the particle detection unit 103a detects particles with a diameter of 0.1 to 0.3 μm. Therefore, an aperture with a cross-sectional area of 0.4 μm^2 (a rectangular one with a width of 1 μm and a height of 0.4 μm, or a circular one with a radius of 0.36 μm) is installed. .In order to detect particles of 2 to 0.8 μm, an aperture cross-sectional area of 2 μm^2 (rectangular with a width of 2 μm and height of 1 μm, or circular with a radius of 0.8 μm) is installed. In the particle detection unit 103b, in order to detect particles with a diameter of 0.4 to 2.0 μm, the aperture cross-sectional area is 14 μm^2 (a rectangular one with a width of 3.5 μm and a height of 4 μm, or a circular shape with a radius of 2.1 μm). ) may be installed.

In addition, when a flow path using the principle of PFF is used in the particle separation flow path 110, the flow rate set by the liquid feeding section is preferably set between 0.1 μL/hour and 1 mL/hour, and particles containing It is preferable that the flow rate of the fluid 100N without particles is at least twice as large as that of the fluid 100P containing particles. How many times the flow rate of the fluid 100N that does not contain particles is preferably higher than that of the fluid 100P that contains particles depends on the channel width of the constricted channel 16 and the diameter of the particles to be separated. When the diameter of the particles to be separated is 1/4 of the channel width of the constricted channel 16, it is preferable that the flow rate of the fluid 100N without particles is at least three times that of the fluid 100P containing particles. When the diameter of the particles is 1/10 of the channel width of the constricted channel 16, it is preferable that the flow rate of the fluid 100N not containing particles is nine times or more than the flow rate of the fluid 100P containing particles. In other words, if the width of the constricted channel 16 is N times the diameter of particles to be separated based on the principle of PFF, the flow rate of 100N of fluid that does not contain particles will be higher than that of fluid 100P that contains particles. It is preferable that the amount is N-1 times or more. With the above flow rate ratio, the particles to be separated will glide along the wall surface 16a of the constricted flow path, making it possible to separate particles based on the principle of PFF.

Furthermore, in the case where a flow path using the principle of PFF is used in the particle separation flow path 110, it is not necessary to cause all the particles contained in the particle-containing fluid 100P to slide down the narrowed flow path wall surface 16a, and relatively large particles Only particles having a certain diameter may be allowed to slide onto the narrow channel wall surface 16a. For example, in the case of FIG. 11(b), FIG. 12, and FIG. 13, if particles with a maximum particle diameter that is less than the maximum particle diameter that can be detected by the particle detection unit 103a that detects small-diameter particles are allowed to slide toward the constricted channel wall surface 16a, Even if particles smaller than the maximum particle diameter are not aligned on the narrow channel wall surface 16a, the particle detection unit 103a can create their distribution based on the ESZ principle. That is, even if there are particles that cannot be completely separated by PFF, the particle size distribution can be created in the particle detection section, so that the excellent effect of ESZ complementing the separation ability of PFF can be obtained.

Furthermore, even under flow conditions in which only particles larger than the maximum particle size that can be detected by the particle detection unit 103a that detects small particle size particles can be allowed to slide toward the narrowed channel wall surface 16a, the HDF theory described above can be applied. Based on this, by setting the flow path resistance so that particles with a particle size that would block the aperture of the particle detection unit 103a do not flow in, it becomes possible to measure a sample having a wide particle size distribution, which is the object of the present invention. In other words, by separating medium and large particles using PFF, separating small particles using HDF, and creating a precise particle size distribution using ESZ, it is possible to measure samples with a wide range of particle size distributions. An excellent effect can be obtained. At this time, since the enlarged channel 17 in the PFF also plays the role of the aforementioned particle diffusion channel 110B, an excellent effect of promoting the separation of small diameter particles having a large diffusion distance per unit time can be obtained. . Furthermore, in this embodiment, small-sized particles also flow into the particle detection section 103c, and in some cases also flow into the particle detection section 102b, so that the small-sized particles flowing into the particle detection section 103a include a particle sample. It becomes part of the fluid 100P. If the length of the enlarged channel 17, which plays the role of the particle diffusion channel 110B, is a sufficient distance for small-sized particles to diffuse, the inflow amount is determined between the particle recovery channel 102a and the channel downstream thereof. Since it is proportional to the flow rate calculated from the structure, it can be quantified from the calculated value. Here, the "distance sufficient for small diameter particles to diffuse" in the length of the enlarged channel 17 means that the sum of the flow rate of the fluid 100N that does not contain particles and the flow rate of the fluid 100P that contains particles is 0.1 μL. If it is in the range of /hour to 1mL/hour, it is preferably 1 μm or more, more preferably 100 μm or more.

In the separation method used in the particle separation channel 110, when using the principle of particle separation using DLD (deterministic lateral displacement method), Dean force, or inertial force, at least one particle introduction channel 101 is required, In order to perform more stable separation, a plurality of particles may be introduced, a sample may be introduced from one of them, and a fluid containing no particles may be introduced from the remaining particle introduction channel 101.

In the separation method used in the particle separation channel 110, when particle separation is performed using an electric field, it is preferable to have a channel structure in which a pair of electrodes are arranged in the separation channel in a direction perpendicular to the flow of the fluid. At this time, not only the size of the particles but also the surface charge in the fluid is a factor in separation, so the particle detection section 103 in or downstream of the particle collection channel 102 must be designed with this in mind. be.

In the separation method used in the particle separation channel 110, when particle separation is performed using a magnetic field, a pair of magnetic field generating components or a magnetic field generator is installed in or outside the separation channel in a direction perpendicular to the fluid flow. It is preferable to have a configuration in which the At this time, not only the size of the particles but also the magnetism of the particles is a factor in separation, so the particle detection section 103 in or downstream of the particle collection channel 102 needs to be designed with this in mind.

In the separation method used in the particle separation channel 110, when particle separation is performed by surface acoustic waves or acoustophoresis, a pair of acoustic transducers are installed in or outside the separation channel in a direction perpendicular to the fluid flow. It is preferable to have a channel structure in which the channels are arranged. At this time, not only the size of the particles but also the rigidity and density of the particles are factors in separation, so the particle detection section 103 within the particle recovery channel 102 or downstream thereof needs to be designed with this in mind. .

The particle detection channel 62 can also have a structure based on principles other than ESZ, such as nanotracking analysis, DLS, scattered light detection, laser diffraction scattering method, resonance mass measurement method, light shielding method, and microscopic observation. method, flow cytometry can be used.

In addition, the present invention detects particles, aggregates, aggregates, and fusions in nanoparticle medicines generated during the formulation process, thereby determining the particle size distribution, average particle size, mode size, intermediate particle size, and It is possible to measure at least one parameter among volume ratio, concentration, number, integrated concentration, and integrated number, or a change over time of the parameter. Based on these measurement results, it is possible to evaluate the stability, safety, and physical properties related to aggregation, association, and fusion of nanoparticle drugs. In addition, by intentionally applying stress such as heating, pressurization, stirring, vibration, changing the suspension solution composition, and freezing/thawing to nanoparticle drugs, it is possible to intentionally create aggregates, aggregates, and fusion of particles in nanoparticle drugs. The stability, safety, and physical properties related to aggregation, association, and fusion of nanoparticle drugs may be evaluated.

Changes over time include changes in the particle size, volume, or number of nanoparticle drugs or their candidate substances over time, as well as changes in the formulation process of nanoparticle drugs or the manufacturing process of their candidate substances. The above-mentioned changes before and after the treatment performed are also included.

EXAMPLES Below, embodiments of the present invention will be described in more detail with reference to Examples. Of course, the present invention is not limited to the following embodiments, and it goes without saying that various modifications can be made to the details. Furthermore, the present invention is not limited to the embodiments described above, and various changes can be made within the scope of the claims, and the present invention also includes embodiments obtained by appropriately combining the disclosed technical means. falls within the technical scope of the invention. Additionally, all documents mentioned herein are incorporated by reference.

Manufacturing of particle detection device A microchip 10 shown in FIG. 15 was manufactured using general photolithography and soft lithography techniques. The specific steps are shown below.
A photoresist SU-8 3005 (Microchem) was dropped onto a 4-inch bare silicon wafer (Filtech Co., Ltd.), and a photoresist thin film was formed using a spin coater (MIKASA). At this time, a diluent Cyclopentanone (Tokyo Ohka Kogyo Co., Ltd.) was added to SU-8 3005 depending on the desired film thickness. Next, a channel pattern is formed on the photoresist film using a mask aligner (Ushio Inc.) and a chrome mask with an arbitrary pattern formed thereon, and the channel pattern is developed using SU-8 Developer (Microchem). By doing this, we created a mold for the flow path we wanted to use.
Subsequently, uncured LSR7070FC (Momentive Performance) was poured into the prepared mold and heated at 80° C. for 2 hours to produce polydimethylsiloxane (PDMS) with the shape of the flow path transferred thereto. The cured PDMS was carefully peeled off from the mold, and after being shaped into an arbitrary size using a cutter, an inlet and an outlet for a flow path were formed using a puncher. After surface-treating the peeled PDMS and slide glass (Matsunami Glass Co., Ltd.) using an oxygen plasma generator (Meiwaforsys Co., Ltd.), the microchip 10 was produced by bonding the PDMS and the slide glass together.
The height of the channel 13 is all 5.5 μm except for the particle detection section 103a and the particle detection section 103c, and the ends of the channel 13 are provided with inlets 14a, 14b and outlets 104a, 104a', 104b that penetrate the top surface of the substrate 11. , 104b', 104c, 104c', and 23 (each with a hole diameter of 2 mm) were provided. The flow path 13 includes a branch flow path 18a (width 20 μm, length 1.5 mm), a branch flow path 18b (width 40 μm, length 500 μm), a narrowed flow path 16 (width 6 μm, length 20 μm), and an enlarged flow path 17. (135 degree expansion angle, maximum expansion channel width 600 μm, length 0.5 mm), drain channel 22 (width 500 μm, length 1.7 mm), particle collection channel 102a (width 75 μm, length 4 mm), particles A recovery channel 102c (width 140 μm, length 7.5 mm) and a particle recovery channel 102b (width 512 μm, length 3.75 mm) were used. The two apertures of the particle detection unit 102a are both 1.6 μm wide, 0.6 μm high, and 2.5 μm long, and the two apertures of the particle detection unit 102c are both 2.6 μm wide and 2.6 μm high. The particle detection unit 102b has two apertures each having a width of 5.8 μm, a height of 5.5 μm, and a length of 10 μm. The shape of each particle detection part is the same as that in FIG. 14, and the k value calculated in equation (1) is calculated from the aperture resistance and the resistance ratio of the flow path from the aperture to the outlet where the electrode is inserted. It was set as .0.

Electrical Detection Example The fabricated microchip 10 was placed on a substrate, and electrodes were connected to a plurality of particle detection units 103 within the microchip 10. The electrodes are composed of a pair of platinum wires, and one electrode is connected to a programmable current amplifier CA5350 (NF Circuit Co., Ltd.) via a conductive wire, and then connected to a PC via an AD converter to receive the transmitted digital signal. Analyzed by LabView. The other electrode connected to the particle detection unit 103 was connected to a 9V dry battery via a conductive wire.
Each inlet was connected to a pressure pump P-PumpBasic (Dolomite) via a Teflon tube, and liquid was fed at a constant flow rate.

Production of large particle detection device A microchip for large particle detection was fabricated in the same manner as the particle detection device described above, except that the pattern of the chrome mask and the conditions for forming a thin film using a spin coater were different. At this time, the height of the channel 13 is designed to be 15 μm except for the particle detection section 103a and the particle detection section 103c. Angle 135 degrees, maximum expansion channel width 570 μm, length 1.25 mm), drain channel 22 (width 545 μm, length 1.4 mm), particle recovery channel 102a (width 75 μm, length 4 mm), particle recovery flow A channel 102c (width 140 μm, length 7.5 mm) and a particle recovery channel 102b (width 350 μm, length 3.7 mm) were used. The two apertures of the particle detection unit 102a are both 2 μm wide, 2.5 μm high, and 10 μm long, and the two apertures of the particle detection unit 102c are both 4 μm wide, 5 μm high, and 10 μm long. The two apertures of the particle detection unit 102b were both 15 μm wide, 15 μm high, and 10 μm long.

Sample preparation (1) Untreated sample Foreign matter was removed from 0.3 μm diameter liposomes (Katayama Chemical Industries) using a syringe filter with a pore size of 0.1 μm (manufactured by Merck Millipore), 0.05% (v/v). ) It was diluted 10,000 times with a 1× PBS solution (phosphate buffer) containing Tween 20.
(2) Treated sample The above-mentioned 0.3 μm diameter liposome was diluted 100 times with the above-mentioned 1× PBS solution containing 0.05% (v/v) Tween 20, and then autoclaved (high pressure steam sterilized) at 121°C. , 2 atm, 20 minutes) and further diluted 100 times with a 1× PBS solution containing 0.05% (v/v) Tween 20.
(3) Untreated antibody drug A 1× PBS solution containing 0.05% (v/v) Tween 20 (phosphate buffered Commercially available antibody drugs A, B, and C were each diluted with 1 mg/mL solution.
(4) Heat-treated antibody drug for verifying changes over time Antibody drug C was heat-treated at 60°C for 30 minutes in the form of a preparation, left at room temperature for 10 minutes, and then treated with 0.05% (v/v) Tween 20. It was diluted to a concentration of 1 mg/mL with a 1× PBS solution containing the sample, and the dilution time was set as 0 minutes after the treatment.
(5) Heat-treated antibody drugs for comparison of antibody aggregate amounts Antibody drugs A, B, and C each have a concentration of 1 mg/mL in a 1× PBS solution containing 0.05% (v/v) Tween 20. The mixture was diluted and heat treated at 68°C for 30 minutes.
(6) Antibody drugs for differential scanning calorimetry measurement Antibody drugs A, B, and C were each dialyzed for 15 hours in a 1×PBS solution containing 0.05% (v/v) Tween 20.
(7) Untreated liposome preparation Ambisome (registered trademark, Sumitomo Dainippon Pharma) was prepared according to the protocol specified by the manufacturer, and then diluted 100 times with a 1x PBS solution containing 0.05% (v/v) Tween 20. did.
(8) Storage liposome formulation The above-mentioned untreated liposome formulation was stored at 40°C for 10 days.

(Example 1)
Using the above-mentioned microchip 10, the prepared processed sample was delivered to the inlet 14a at a flow rate of 2.4 μL/hour, and 10 μL of 1×PBS containing 0.05% (v/v) Tween 20 was delivered to the inlet 14b. The liquid was fed at a flow rate of /hour. Next, the particles flowing into each of the three particle collection channels 102 were detected for one minute based on the electrical detection example described above, and the measurement results were summarized into one histogram as shown in FIG. 16. At this time, the liposome was converted into a weight concentration (μg/mL) as a true sphere with a specific gravity of 1.05 μg/mL, and summarized as a histogram.
An experiment was conducted in the same manner for the untreated sample, and the measurement results obtained were summarized into one histogram as shown in FIG. 17.
Comparing the two, a distribution of large particles that appear to be aggregates or fusions was observed only in the treated sample in an area of 0.5 μm or more, confirming that liposome aggregates or fusions were generated by the autoclave treatment. did it.

(Comparative example 1)
The treated sample was measured using AggregatesSizer (Shimadzu Corporation), and the measurement results shown in FIG. 18 were obtained. Further, the untreated sample was also measured in the same manner, and the measurement results shown in FIG. 19 were obtained. The measurement principle of AggregatesSizer is the laser diffraction/scattering method, which irradiates a particle group with a laser beam and calculates the particle size distribution from the intensity distribution pattern of the diffracted/scattered light emitted from the particle group. No separation was performed.
Comparing the two, there was no significant difference in particle size distribution with a diameter of 0.5 μm or more, which confirmed that it is difficult to detect a small amount of liposome aggregates or fusions using this method.

(Example 2)
Using the above-mentioned microchip 10, the heat-treated antibody drug (0 minutes after treatment) prepared for verifying changes over time was delivered to the inlet 14a at a flow rate of 2.4 μL/hour, and 0.05% (0.05%) was delivered to the inlet 14b. v/v) 1×PBS containing Tween 20 was delivered at a flow rate of 10 μL/hour. Next, the particles flowing into each of the three particle collection channels 102 were detected for one minute based on the electrical detection example described above, and the measurement results were summarized into one histogram as shown in FIG. 20(a). At this time, the antibody aggregate was converted into a weight concentration (μg/mL) as a true sphere with a specific gravity of 1.32 μg/mL, and summarized as a histogram. Furthermore, when the heat-treated antibody drugs were similarly measured 10 minutes and 120 minutes after treatment, the results were shown in Figures 20(b) and 20(c), and the amount of antibody aggregates and peak top position changed over time. I observed how it was increasing.
Furthermore, when experiments were conducted in the same manner with untreated antibody drugs, almost no particles were detected, and it was confirmed that there were almost no antibody aggregates or impurities in the untreated drugs.

(Comparative example 2)
Samples of heat-treated antibody drugs for time-dependent change verification at 0 minutes, 10 minutes, and 120 minutes after treatment were measured in the same manner as in Comparative Example 1, and the measurement results shown in Figures 21(a) to (c) were obtained. Obtained. From this, we confirmed that it is also possible to observe changes in antibody aggregates over time using the laser/diffraction scattering method. However, the peak width is broader than in Example 2, making quantitative measurement difficult.

(Example 3)
Using the above-mentioned microchip 10, heat-treated antibody drugs A, B, and C for comparison of the amount of antibody aggregates prepared are sent to the inlet 14a at a flow rate of 2.4 μL/hour, and 0.05% to the inlet 14b. (v/v) 1×PBS containing Tween 20 was delivered at a flow rate of 10 μL/hour. Next, based on the electrical detection example described above, particles flowing into each of the three particle collection channels 102 are detected for one minute, and the measurement results are summarized into one histogram as shown in FIGS. 22(a) to 22(c). became. At this time, the antibody aggregate was converted into a weight concentration (μg/mL) as a true sphere with a specific gravity of 1.32 μg/mL and summarized as a histogram. In addition, the integrated amounts of antibody aggregates were 7.3 μg/mL for antibody drug A, 2.1 μg/mL for antibody drug B, and 0.74 μg/mL for antibody drug C.
As shown in Figure 22, it was confirmed that the distribution and amount of generated antibody aggregates differed depending on the type of antibody drug.

(Comparative example 3)
Heat-treated antibody drugs A, B, and C for comparison of antibody aggregate amounts were measured in the same manner as in Comparative Example 1, and the measurement results shown in FIG. 23 were obtained. From this, although it is possible to confirm differences in the distribution and amount of antibody aggregates in different antibody drugs using the laser/diffraction scattering method, it is not possible to detect antibody aggregates with respect to antibody drug C in Figure 23(c), making accurate measurement difficult. was confirmed to be difficult.

(Reference example 1)
FIG. 24 shows the results of measuring the thermal stability of each antibody for differential scanning calorimeter measurement antibody drugs A, B, and C using a differential scanning calorimeter MicroCal (Malvern). The positions of the endothermic peak tops of each are 72.3°C for antibody drug A, 72.8°C for antibody drug B, and 75.0°C for antibody drug C, and the thermal stability is highest for antibody drug C, followed by antibody drug B. The results indicate that antibody drug C has the highest stability and the lowest stability.
From the results of Example 3 and Reference Example 1, it was confirmed that the lower the thermal stability of the antibody drug, the higher the amount of antibody aggregates, and it was confirmed that the thermal stability and the amount of antibody aggregates were correlated. . Therefore, we have found that it is possible to evaluate the stability of antibody drugs using the present invention.

(Example 4)
Using the above-described microchip 10, the prepared untreated liposome preparation was delivered to the inlet 14a at a flow rate of 3.6 μL/hour, and 1× PBS containing 0.05% (v/v) Tween 20 was delivered to the inlet 14b. The liquid was pumped at a pressure of 800 mbar. Subsequently, based on the electrical detection example described above, the particles flowing into the particle recovery channel 102a are detected for 2 minutes, the particles flowing into the particle recovery channel 102c are detected for 4 minutes, and the particles flowing into the particle recovery channel 102b are detected for 100 seconds. Detected.
In addition, using a microchip for detecting large particles, the prepared untreated liposome preparation was sent to the inlet 14a at a flow rate of 16 μL/hour, and 1×PBS containing 0.05% (v/v) Tween 20 was sent to the inlet 14b. was pumped at a pressure of 400 mbar. Subsequently, based on the electrical detection example described above, the particles flowing into the particle collection channel 102a are detected for 2 minutes, the particles flowing into the particle recovery channel 102c are detected for 4 minutes, and the particles flowing into the particle recovery channel 102b are detected for 4 minutes. Detected.
The measurement results of particles with a diameter of less than 0.5 μm detected with the microchip 10 and particles with a diameter of 0.5 μm or more detected with the large particle detection microchip are summarized in a histogram as shown in FIG. 25(a). At this time, the liposome was converted into a weight concentration (μg/mL) as a true sphere with a specific gravity of 1.05 μg/mL, and summarized as a histogram.
As shown in FIG. 25(a), the untreated liposome preparation has a wide distribution from several tens of nanometers to several micrometers, so in addition to the microchip 10, a large particle detection microchip that can measure larger particles is used in combination. By using the measurement results with the microchip 10 for particles less than 0.5 μm and the measurement results with the large particle detection microchip for particles larger than 0.5 μm, the histogram shown in FIG. 25(a) can be created. Obtained.
Furthermore, the stored liposome preparation was measured in the same manner, and the histogram shown in FIG. 25(b) was obtained.
Comparing the two, distribution of large particles that appear to be aggregates or fusions in the region of 0.5 μm or more is seen in both cases, and furthermore, in the stored liposome preparations, the amount of aggregates or fusions ranges from 0.6 μg/mL to 0.6 μg/mL. It was confirmed that the concentration had increased to 1.2 μg/mL.

(Comparative example 4)
Untreated liposome preparations and stored liposome preparations were measured using a dynamic light scattering method (Otsuka Electronics, Zeta potential/particle size measurement system), and the measurement results shown in Figures 26(a) and (b) were obtained. . Note that the dynamic light scattering method is a method in which a group of particles is irradiated with a laser beam and the particle size distribution is calculated from the temporal change in the intensity of the scattered light emitted from the group, and particles are not separated according to their size. Not yet. In addition, dynamic light scattering is a method commonly used to measure the particle size of liposome formulations, and it is also listed as the main particle size distribution measurement method in the guidelines for the development of liposome formulations published by the Ministry of Health, Labor and Welfare. This was used as a comparative example.
In addition, from FIG. 26, it was confirmed that it was difficult to detect the aggregates or fusions since the distribution of large particles that appeared to be aggregates or fusions was not observed in the dynamic light scattering method.

10 Microchip 11 Substrate 13 Channel 14, 14a, 14b Inlet 16 Constricted channel 16a Sample liquid side narrowed channel wall 16b Sheath liquid side narrowed channel wall 17 Enlarged channel 17a Sample liquid side enlarged channel wall 17b Sheath liquid side Narrow expansion channel wall surface 18a, 18b Entrance side branch channel 19 Enlargement start point 21 Region 22 Drain channel 23 Outlet 24a, 24b Angle 40 Slope portion 50 Particle 51 Particle flow direction 52 Aperture forming structure 53 Aperture 54, 54a, 54b Electrode 55 Lead wire 56 Electrical measuring device 57 Power supply 58 Conductive solution 59 Electrode insertion port 60 Relay channel 61 Analysis section 62 Particle detection channel 100 Fluid 100P Fluid 100N Fluid 101 Particle introduction channel 102a-c Particle collection channel 103a-c Particle Detection section 104a-c Outlet 110 Particle separation channel 110A Branch section 110B Particle diffusion channel

Claims (5)

A method for detecting particles contained in nanoparticle pharmaceuticals, the method comprising:
Separates particles in the direction perpendicular to the fluid flow according to their particle size,
dividing the separated particles into two or more channels,
detecting the particles with an electric detector including electrodes placed across an aperture installed in the flow path ;
The method described above, characterized in that the range of particle diameters that can be detected by the electric detector differs depending on the cross-sectional area of the aperture . 2. The method according to claim 1 , wherein part of the particle size ranges that can be detected by the electric detectors overlap. By adjusting at least one parameter among the number of channels, the shape, width, height, and length of the branch part, and creating a channel structure that does not allow particles of a certain size or more to mix in, particles can be The method according to claim 1 or 2 , characterized in that the flow path is divided into a plurality of channels. The method according to any one of claims 1 to 3 , characterized in that the particles are separated using the principle of pinched flow fractionation. Detecting at least one of particles, aggregates, aggregates, and fusions in nanoparticle pharmaceuticals by the method according to any one of claims 1 to 4 ,
A method for measuring at least one parameter among particle size distribution, average particle size, mode diameter, intermediate particle size, volume ratio of each particle, concentration, number, cumulative concentration, and cumulative number, or a change over time in the parameter.