Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/16—Blood plasma; Blood serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D7/00—Sublimation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
  • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
  • B01J2/04—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops in a gaseous medium
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F26—DRYING
  • F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
  • F26B17/00—Machines or apparatus for drying materials in loose, plastic, or fluidised form, e.g. granules, staple fibres, with progressive movement
  • F26B17/10—Machines or apparatus for drying materials in loose, plastic, or fluidised form, e.g. granules, staple fibres, with progressive movement with movement performed by fluid currents, e.g. issuing from a nozzle, e.g. pneumatic, flash, vortex or entrainment dryers
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F26—DRYING
  • F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
  • F26B5/00—Drying solid materials or objects by processes not involving the application of heat
  • F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
  • F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing

Definitions

• the present invention relates to dried plasma that has excellent particle homogeneity and maintains biological activity, and a method for producing the same.
• the freeze-vacuum drying method is a useful method because it is theoretically possible to scale up and maintain the activity of various components, but when applied to plasma, it is difficult to use the conventional equipment conditions because it contains many types of protein components. Due to the characteristic of plasma that the self-freezing rate of each component is different, the production of high-quality dried plasma on an industrial scale is difficult due to the non-uniformity of the particles of the obtained dried plasma and the occurrence of unfrozen clumps. There were factors that prevented it. As described above, conventional methods for producing dried plasma have had the problem of not being able to provide medical facilities with practical dried plasma that has excellent particle homogeneity and maintains the biological activity of each plasma component. .
• the present inventors have intensively investigated the possibility of pulverizing plasma using the freeze-vacuum drying method, which in principle can generate frozen microparticles without applying a heat load during spraying, and have designed a freeze-vacuum dryer and By optimizing the manufacturing conditions to produce dried plasma, we have been able to obtain highly useful dried plasma with excellent particle homogeneity and maintaining the biological activity of each plasma component.
• the inventors also discovered that plasma has high solubility when reconstituted, and that the number of insoluble particles that pose a risk of side effects is reduced, leading to the completion of the present invention.
• Specific embodiments of the present invention are as follows. However, it is not limited to these.
• [About the dried plasma of the present invention] [1] Dried plasma, which has a D50 of 200 ⁇ m to 400 ⁇ m based on a volume-based particle size distribution measured by a laser diffraction scattering particle size distribution measurement method. [2] The value of (D90-D10)/D50 is 0.1 to 2.0 in D10, D50, and D90 according to the volume-based particle size distribution measured by laser diffraction scattering particle size distribution measuring method.
• the dried plasma according to the above [1] characterized in that: [3] The dried plasma according to [2] above, wherein the value of (D90-D10)/D50 is 0.3 to 2.0.
• the content of coarse particles of 1000 ⁇ m or more is 30% by weight or less of the dry plasma weight (preferably 0 to 25% by weight, more preferably 0.1 to 20% by weight), [1] above. ] to [3].
• the dried plasma according to any one of [3].
• the activity recovery rate of ristocetin cofactor activity is 70% or more (more preferably , 70 to 95%), the dried plasma according to any one of [1] to [7] above.
• Fibrinogen activity is 150 to 400 mg/dL (preferably 200 to 400 mg/dL) in plasma reconstituted by dissolving dried plasma in 20 mmol/L Glycine (pH 2.4). The dried plasma according to any one of [1] to [8].
• the activity of Factor V is 0.5 IU/mL (preferably 0.9 IU/mL) or more. (The activity is preferably 1.4 IU/mL or less), the dried plasma according to any one of [1] to [9] above.
• Protein C activity is 0.7 IU/mL (preferably 1.1 IU/mL) or more in plasma reconstituted by dissolving dried plasma in 20 mmol/L Glycine (pH 2.4). (The activity is preferably 1.4 IU/mL or less), the dried plasma according to any one of [1] to [10] above.
• the FVIII activity is 0.5 IU/mL or more (the activity is preferably 1.5 IU/mL or more). /mL or less), the dried plasma according to any one of [1] to [11] above.
• the activity of FXI is 0.5 IU/mL or more (the activity is preferably 1.5 IU/mL or more). /mL or less), the dried plasma according to any one of [1] to [12] above.
• the ⁇ 2-antiplasmin activity is 0.2 IU/mL or more (the activity is preferably 1 .1 IU/mL or less), the dried plasma according to any one of [1] to [13] above.
• the number of insoluble fine particles with a size of 10 ⁇ m or more is 2400 or less, [1] to [14] above. Dried plasma as described in .
• the plasma is spouted into the freeze-vacuum drying tower under vacuum from a nozzle that is provided at the upper end of the freeze-vacuum drying tower and has a pore diameter of 50 to 350 ⁇ m for spouting the plasma. a step of self-freezing the microscopic raw material liquid particles formed by the process to form microfrozen particles, and a step of capturing the microscopic frozen particles on a capture surface in the freeze-vacuum drying tower and heating and sublimation drying. Method for producing plasma. [17] The production method according to [16] above, wherein the pore diameter for ejecting plasma is 100 to 250 ⁇ m.
• the present invention provides highly useful dried plasma that has excellent particle homogeneity and maintains the biological activity of each plasma component, and also has high solubility during plasma reconstitution, which poses no risk of side effects. Dried plasma that also has a reduced number of insoluble particulates, and methods for producing the same, are provided.
• FIG. 1 is a schematic cross-sectional view of a freeze-vacuum drying apparatus 1 used in Examples.
• FIG. 2 is a schematic cross-sectional view of the nozzle used in the example.
• FIG. 3 is a schematic diagram of the Thrombin Generation Assay using the CAT system used to measure the amount of thrombin generated in the Examples.
• FIG. 4 is a diagram showing the particle size distribution of Example 4 plasma and Comparative Example plasma examined in Examples.
• FIG. 5 is a diagram showing the weight particle size distribution of Example 4 plasma and Comparative Example plasma examined by sieving test in Example.
• FIG. 6 is a diagram schematically showing parameters of ROTEM measurement.
• the dried plasma of the present invention (also referred to as “this dried plasma”) is: "Dried plasma characterized by having a D50 of 200 ⁇ m to 400 ⁇ m based on a volume-based particle size distribution measured by a laser diffraction scattering particle size distribution measuring method.” It is. Laser diffraction scattering particle size distribution measuring method is a method widely used in this technical field to evaluate the particle size distribution (particle size distribution) of powders.
• the measurement method of "particle size analysis - laser diffraction/scattering method" specified in standard number JIS Z8825 is used, and measurement can be performed using a commercially available measurement device (for example, Microtrack MT3100 (NIKKISO)).
• a commercially available measurement device for example, Microtrack MT3100 (NIKKISO)
• the patterns of diffracted and scattered light vary depending on the size of the particles, and this is used to detect particle size information from the pattern of the diffracted and scattered light, and calculate the particle size distribution through arithmetic processing.
• the measurement was performed by irradiating the sample with laser light while dispersing the powder sample with air, and measuring the particle size distribution from the diffraction scattering pattern (dry laser diffraction scattering method).
• D10 10% diameter
• D50 50% diameter, median diameter
• D90 90% diameter
• this dried plasma is characterized by having a D50 in the range of 200 ⁇ m to 400 ⁇ m. Powder having a particle diameter D50 of 200 ⁇ m to 400 ⁇ m has good powder handling properties, so the proportion of coarse particles contained in the powder is reduced.
• the value calculated by (D90-D10)/D50 is within the range of 0.1 to 2.0 (more preferably is within the range of 0.3 to 2.0). The closer this value is to 1, the higher the homogeneity of the particles constituting the powder.
• This dried plasma has extremely high homogeneity of the particles constituting the powder by falling within the above numerical range. As will be described later, the activity (eg, fibrinogen activity) of the dried plasma is improved due to the high homogeneity of the particles.
• the coarse particle content rate of 1000 ⁇ m or more is 30% by weight or less (preferably 20% by weight or less) of the dry plasma weight.
• the coarse particle content is determined by subjecting the dried plasma to be evaluated to a sieving test using a mesh (having openings according to the intended classification, such as 1000, 500, 300 ⁇ m, etc.). It can be determined by measuring the weight of the residual powder on the mesh after classification and examining the weight particle size distribution (for more specific measurement conditions, see the description in Example 2 below).
• the coarse particles of 1000 ⁇ m or more are suppressed within the above range, thereby eliminating the need for a classification operation in the manufacturing process and improving the recovery rate of dried plasma in the manufacturing process.
• This dried plasma has the following properties.
• (1) Dissolution characteristics The dissolution time of dried plasma in a 20 mmol/L Glycine (pH 2.4) solution is within 5 minutes (or within 4 minutes).
• the dried plasma was dissolved (plasma reconstitution) by stirring with hand shaking, and dissolved at a weight ratio of 10 w/v% under room temperature conditions (see the description in Example 2 below).
• FFP fresh frozen plasma
• FVIII activity is 0.5 IU/mL or more.
• FXI activity is 0.5 IU/mL or more.
• ⁇ 2-antiplasmin activity is 0.2 IU/mL or more.
• the "standard value of fibrinogen” was based on the standard value of "Octaplas LG (registered trademark)" (Octapharma), an SD-treated plasma preparation.
• Octaplas LG registered trademark
• the present dried plasma is highly useful after reconstitution.
• the specific method and conditions for measuring the activity of each blood coagulation factor are as detailed in Example 2 below.
• the number of insoluble fine particles in the plasma solution after plasma reconstitution is most affected by the clarity of the drug substance at the time of manufacturing the dried plasma; In some cases, it may be necessary to comprehensively judge the evaluation results for dried plasma obtained from multiple manufacturing lots.
• this dried plasma has both excellent powder properties and excellent biological activity.In addition, it has excellent powder particle homogeneity, high solubility during plasma reconstitution, and is highly soluble in various blood coagulation factors. It has the characteristics of maintaining its activity and reducing the number of insoluble particles that pose a risk of side effects, making it extremely useful in medical practice. An example of a specific usage mode is described below. “Since this dried plasma is in the form of a powder, it can be sealed in a plastic bag, does not require frozen storage, and is an easily portable preparation, so it can be used in mobile medical settings such as doctor helicopters.
• This dried plasma can be used as a reconstitution solution using a 20 mmol/L glycine solution with a pH of 2.4, and the plasma in the bag is dissolved by adding the reconstitution solution to the powder so that the weight ratio is 10 w/v%.
• Reconstituted plasma preparations are administered intravenously with red blood cells and plasma mixed at a ratio of 1:1 or 1:2 at an administration rate of 10 to 15 mL/kg for coagulation factor replacement in cases of massive bleeding, etc., and the amount used is Approximately 4 units of plasma (one unit is 200 mL) is administered.
• plasma preparations are administered at 40 to 60 mL/kg, and the amount used is approximately 1 to 1.5 times the patient's circulating plasma volume. Make an exchange. ”
• the dried plasma can be produced by the following production method using a freeze-vacuum dryer. "It is formed by ejecting plasma into the freeze-vacuum drying tower under vacuum from a nozzle with a pore diameter of 50 to 350 ⁇ m for spouting plasma, which is installed at the upper end of the freeze-vacuum drying tower.
• a process for producing dried plasma which comprises the steps of: self-freezing the microscopic raw material liquid particles to produce micro-frozen particles; and capturing the micro-frozen particles on a capture surface in the freeze-vacuum drying tower, heating and sublimation drying.
• freeze-vacuum drying is a drying method used in this technical field as a method for drying substances dissolved or dispersed in a raw material liquid and extracting them as fine powder. It is one of the laws. There are some substances that are denatured by heating and lose their physiological activity, and others that impair their flavor. For these, the freeze-vacuum drying method is adopted.
• a known method is to self-freeze raw material liquid particles to produce micro-frozen particles, heat the micro-frozen particles under vacuum at low temperature, sublimate the frozen solvent or dispersion medium, and dry them to extract the micro-frozen particles as a fine powder. (For example, see Patent Document 1).
• Plasma obtained by thawing fresh frozen plasma can be used as plasma as a raw material.
• Fresh Frozen Plasma also written as "FFP” is plasma obtained from blood donation that is immediately frozen to below -40°C within 6 hours of collection, and is sold by the Japanese Red Cross Blood Center. There is.
• virus inactivation treatment When dried plasma powder is used by returning it to plasma, it is preferable to perform virus inactivation treatment in advance, and for example, it is preferable to use S/D-treated plasma (also referred to as "SDP").
• S/D treatment of plasma is an abbreviation for solvent (organic solvent)/detergent (surfactant), and is one of the methods to eliminate the infectivity of viruses with lipid membranes, and is widely used in this technical field.
• TNBP tri-n-butyl phosphate: organic solvent
• Triton X-100 polyethylene glycol mono-p isooctyl phenyl ether: surfactant
• Tween 80 surfactant
• oil is added to the S/D-treated plasma solution, TNBP is separated into an oil layer, the aqueous layer is collected, and after filtration, a surfactant such as Triton X-100 is removed by reverse phase chromatography. It can be used after filtering the bacteria.
• S/D treatment of plasma is an extremely important step from the perspective of ensuring the safety of plasma preparations, but since blood coagulation factors in plasma are deactivated by S/D treatment, such activities may be weakened.
• S/D-treated plasma As a raw material, there has been a long-awaited pulverization technology that reduces further deactivation due to pulverization and allows mass processing in a highly active state. I've been The present invention meets these demands.
• freeze-vacuum drying apparatus and dried plasma manufacturing method described here are merely examples, and the specific configuration of the present invention is not limited to this embodiment, but may be within the scope of the spirit of the present invention. This includes design changes, etc.
• FIG. 1 is a schematic cross-sectional view of a freeze-vacuum dryer ( ⁇ PD400, Y010-0777, ULVAC manufactured by ULVAC Co., Ltd.) (hereinafter also referred to as "freeze-vacuum dryer 1") used in the examples described later.
• the freeze-vacuum drying apparatus 1 is an apparatus for producing fine powder P of dried plasma using plasma as a raw material liquid L.
• the freeze-vacuum drying apparatus 1 diverges the raw material liquid L into fine raw material liquid particles R within its cylindrical freeze-vacuum drying tower 11 .
• the minute raw material liquid particles R self-freeze during the fall to become minute frozen particles Q.
• the falling frozen microparticles Q of dried plasma are captured, collected, and heated at a low temperature to sublimate the ice contained in the frozen microparticles Q, thereby drying the frozen microparticles Q.
• a nozzle 12 is provided at the upper end of the freeze-vacuum drying tower 11 in order to inject the raw material liquid L supplied from a raw material liquid supply container 41 (described later) as a liquid column K.
• a nozzle is used in which the pores 14 of the nozzle 12 have a diameter of 50 to 350 ⁇ m (preferably 100 to 250 ⁇ m, more preferably 200 ⁇ m).
• the dispenser nozzle, straight nozzle, and tapered nozzle shown in FIG. therefore, it is preferable to use a dispenser nozzle.
• the liquid column K is formed to a certain length determined by the conditions for injecting the raw material liquid L, and after splitting at its lower end due to the surface tension of the raw material liquid, it diverges and the minute raw material liquid particles R form into particles of a certain size. It is formed. Then, the microscopic raw material liquid particles R self-freeze during the fall to become microscopic frozen particles Q.
• the bottom of the freeze-vacuum drying tower 11 there is a slope that receives and captures the falling micro-frozen particles Q on a slope, and heats them at a low temperature to sublimate and dry the ice contained in the micro-frozen particles Q.
• a transfer device 18, such as a vibrating feeder, is provided for drying the plasma into a fine powder P of dried plasma. Note that the drying is performed in stages of initial drying, primary drying, and secondary drying, and the temperature and pressure inside the freeze-vacuum drying tower (also referred to as "inside tank pressure”) are gradually changed. Specific conditions are shown in Table 1 in Examples below.
• a jacket 17 (not shown) is provided on the bottom side of the conical-shaped collection/heating device 15, and serves as a circulation path for hot water introduced from the outside of the freeze-vacuum drying tower 11 and returned to the outside.
• the micro frozen particles Q of dried plasma are received by the frozen micro particles Q of dried plasma, and while they slide down the capturing surface 15s, which is the inclined surface of the collection/heating device 15, and fall downward from the opening 16 in the center.
• the frozen microparticles Q are heated at a low temperature to sublimate the ice contained therein.
• the surface may be treated with fluororesin, and the collection/heating device 15 may be subjected to minute vibrations.
• a heater may be embedded in the lower side of the capture surface 15s of the collection/heating device 15 to heat the water.
• a portion 11E extending leftward in FIG. 1 is formed at the bottom of the freeze-vacuum drying tower 11, and a transfer device 18 is installed from the bottom to the extending portion 11E. That is, the transfer device 18 receives the micro frozen particles Q with reduced ice content falling from the opening 16 at the bottom of the collection/heating device 15, and heats and dries them while conveying them to the left in FIG. At the downstream end, a fine powder P of dried dried plasma is obtained.
• a planar heater is attached as a heating means, although not shown.
• the extending portion 11E of the freeze-vacuum drying tower 11 is bent directly below after passing over the downstream end of the transfer device 18, and becomes a hanging path 11F where the fine powder P of dried plasma falls.
• the lower part is inserted into a collection/weighing chamber 21, which will be described later, and the lower end thereof is constricted and an automatic on-off valve 19 is attached.
• the collection/weighing chamber 21 into which the above-mentioned hanging passage 11F is inserted is provided integrally with the freeze-vacuum drying tower 11, and the dried plasma fine powder P falling down the hanging passage 11F is stored in a container 22 and weighed. Ru.
• a sealing/removal chamber 31 is connected to the recovery/weighing chamber 21 via a gate valve 29 .
• the gate valve 39 that opens and closes between the container and the atmosphere is opened and the container is taken out to the atmosphere. It will be done.
• the sealing/removal chamber 31 can be evacuated independently even though it is not shown.
• a conveyor 30 is provided in each of the collection/weighing chamber 21, the sealing/removal chamber 31, and the atmospheric side to transport the container 22, but in particular, the conveyor 30 in the collection/weighing chamber 21 is Reference numeral 30 is attached with a load cell type weight measuring device, although not shown.
• a supply container 41 for the raw material liquid L is installed above the left side of the freeze-vacuum drying tower 11.
• a raw material liquid supply pipe 42 is installed to connect the bottom of the raw material liquid supply container 41 and the nozzle 12 at the upper end of the freeze-vacuum drying tower 11.
• a valve 43 is provided.
• a nitrogen gas pipe 44 for applying supply pressure to the upper space of the raw material liquid L stored in the raw material liquid supply container 41 is arranged together with an on-off valve 45 . Note that the raw material liquid L may be supplied under pressure using a pump.
• a vacuum exhaust system for evacuating the inside of the freeze-vacuum drying tower 11 is provided on the right side of the bottom of the freeze-vacuum drying tower 11. That is, a roots pump 52 and a rotary pump 51 are connected to a vacuum exhaust pipe 57 connected to the bottom of the freeze-vacuum drying tower 11 via a cold trap 53 equipped with a refrigerant pipe 54 from a refrigerator. An exhaust valve 55 is provided between the trap 53 and the roots pump 52, and an on-off valve 56 is provided between the freeze-vacuum drying tower 11 and the cold trap 53.
• the spray-type freeze-vacuum drying apparatus 1 of this embodiment is constructed as described above, and its operation will be explained next.
• a nozzle 12 having a pore 14 with a diameter of 50 to 350 ⁇ m (preferably 100 to 250 ⁇ m, more preferably 200 ⁇ m) as an injection hole for the raw material liquid L is provided at the upper end of the freeze-vacuum drying tower 11. (preferably a dispenser nozzle).
• the raw material liquid supply container 41 stores plasma as the raw material liquid L.
• the automatic on-off valve 19 in the recovery/weighing chamber 21 is opened, the on-off valve 56 and the exhaust valve 55 of the vacuum exhaust pipe 57 are opened, and the rotary pump 51 and Roots pump 52 are used to evacuate the cold trap 53.
• tank pressure 1.0 to 10 Pa.
• hot water whose temperature is adjusted from 10° C. to 30° C. is circulated in the jacket 17 on the lower surface side of the collection/heating device 15 at the bottom of the freeze-vacuum drying tower 11, and the transfer device 18 is also energized to a planar heater (not shown). The temperature was kept at 30°C. At that point, all valves other than those mentioned above are closed.
• a liquid column K of the raw material liquid L is formed downward from the pores 14 of the liquid column K, which splits from the lower end due to surface tension, and the raw material liquid L is dispersed in a mist, and the formed minute raw material liquid particles R are Fall downwards.
• the flow rate (linear flow rate) of the raw material liquid L in the pores 14 of the nozzle 12 is in the range of 8 to 18 m/sec (preferably 9 to 17 m/sec, more preferably 10 to 16 m/sec). Sprayed.
• the formed fine raw material liquid particles R self-freeze as the water evaporates while falling through the freeze-vacuum drying tower 11 under vacuum, and the latent heat of vaporization is taken away, resulting in the formation of fine frozen particles Q of dried plasma. It continues to fall and is captured by the upper inclined capture surface 15s of the collection/heating device 15.
• the frozen microparticles Q of dried plasma are heated by the capture surface 15s of the collection/heating device 15, and the ice contained therein is sublimated and dried, and at the same time, they slide down the slope and are released from the opening 16 at the bottom. It falls onto the transfer machine 18.
• the fine powder P discharged from the downstream end of the transfer device 18 was stored in the container 22 in the recovery/weighing chamber 21 via the hanging path 11F, and was weighed.
• the automatic opening/closing valve 19 is closed, the gate valve 29 is opened, and the container 22 is carried out to the seal removal chamber 31, and a new container 22 is collected and placed in the weighing chamber 21.
• the gate valve 29 is closed, the automatic opening/closing valve 19 is opened, and storage of the plasma fine powder P is resumed.
• the container 22 containing the plasma fine powder P carried out to the sealing/removal chamber 31 was capped with a stopper 23 by the cappering machine 32, and then the gate valve 39 was opened and the container 22 was carried out to the atmosphere. After carrying in a new container 22 to replace it, the gate valve 39 is closed and the chamber is evacuated to achieve the same degree of vacuum as the collection/weighing chamber 21. The above operation is repeated in the recovery/weighing chamber 21 and the sealing/removal chamber 31.
• the collecting/heating device 15 may be modified.
• the lower part may be formed into a non-linear (for example, spiral) passage to heat the passage.
• a freeze-vacuum drying method a raw material liquid is freeze-vacuum-dried in a short time by a heating section in a non-linear path, and dried particles are collected in a collecting section.
• the apparatus is compact and does not require a large exhaust mechanism, making it possible to reduce costs.
• Fresh frozen plasma (FFP) was thawed and the plasma was pooled to form an FFP pool.
• the FFP pool solution was filtered to remove cell debris and debris.
• organic solvents Solvent/Detergent
• TNBP final concentration 1%)
• Triton "S/D processing” was performed.
• the liquid after the S/D treatment was subjected to oil extraction using soybean oil or castor oil, TNBP was transferred to the upper oil layer, the lower aqueous layer was recovered, and this liquid was filtered.
• Triton X-100 can be removed by adsorption to an octadecyl group chemically bonded (C18) ODS column, which is commonly used in reverse phase chromatography. After filtration, the solution was passed through an ODS column to remove Triton X-100.
• the column-passing liquid was collected, filtered through a sterilizing filter, and used as a raw material (SD Plasma drug substance; "SDP").
• Examples 1 to 2 Examination of manufacturing conditions for powdered dried plasma of the present invention
• Example 1 Optimization of nozzle diameter
• Three types of nozzles were used in the freeze-vacuum dryer 1 ( ⁇ PD400, Y010-0777, ULVAC manufactured by ULVAC Co., Ltd.).
• Dried plasma was prepared from SDP using a straight nozzle with diameters of 100 ⁇ m, 200 ⁇ m, and 300 ⁇ m (Examples 1(1) to 1(3)).
• FIG. 2 shows a specific form of the straight nozzle.
• the illustrated nozzle has a diameter of 200 ⁇ m, but those skilled in the art can appropriately obtain other diameters, and commercially available products can also be used.
• Table 1 shows the above study conditions.
• Table 2 shows the dispersibility of dried plasma powder particles prepared under each study condition.
• the particle distribution was measured dry using the laser diffraction scattering particle size distribution measurement method described below.
• JISZ8825 Laser diffraction scattering particle size distribution measurement method (dry method) (Device name) Microtrack MT3100 (NIKKISO) (Analysis conditions) Number of measurements: 1 time Measurement time: 10 seconds
• Particle permeability Transmission Particle refractive index: 1.81
• Particle shape Non-spherical Solvent name: Air Solvent refractive index: 1.00 Measurement range: 0.687-995.6 ⁇ m
• the diameter of the nozzle is preferably 50 to 350 ⁇ m, more preferably 100 to 250 ⁇ m.
• a person skilled in the art can appropriately select the diameter of the nozzle in consideration of the condition of the plasma used (eg, viscosity) and other manufacturing process conditions.
• Example 2 Optimization of nozzle shape and device parameters [Summary]
• spray nozzles dispenser nozzle, straight nozzle, and tapered nozzle; the specific configuration of each nozzle is shown in FIG. 2; the diameter of the illustrated nozzle is 200 ⁇ m.
• FIG. 2 the specific configuration of each nozzle is shown in FIG. 2; the diameter of the illustrated nozzle is 200 ⁇ m.
• those skilled in the art will be able to obtain other calibers as appropriate, and it is also possible to use commercially available products.
• Dried plasma was prepared by adjusting the pressure inside the tank during drying and the linear flow rate (Examples 2(1) to 2(9)).
• the powder characteristics particle size distribution, coarse particle content
• the dispenser nozzle was the most suitable nozzle for both inhibiting the formation of unfrozen clots and maintaining blood coagulation activity.
• the results showed that coarse particle formation was suppressed and the activity recovery rate was high by spraying at a spray pressure of 0.05 to 0.075 MPa (linear flow velocity of 10 to 14 m/sec). Obtained.
• Example 2 (5) is a retest of (4)
• Example 2 (7) is a retest of (6), and the lots of SDP are different.
• the dispersibility of dried plasma particles was evaluated using (D90-D10)/D50.
• (D90-D10)/D50 is around 1.0, the standard deviation of the D50 particle size becomes small and the homogeneity (monodispersity) of the particle size becomes high.
• the larger the value of (D90-D10)/D50 the larger the width of the standard deviation of the D50 particle diameter, resulting in non-uniform particles.
• the dispenser nozzle and the tapered nozzle had a smaller value of (D90-D10)/D50 and higher monodispersity.
• the nozzle with the best particle homogeneity was the dispenser nozzle.
• APTT Activated partial thromboplastin time
• PT prothrombin time
• Fibrinogen activity mg/dL
• Factor VIII activity IU/mL
• Factor V activity IU/mL
• Factor XI activity IU/mL
• Clotting time method Clotting time method
• vWF ristocetin activity (vWF:Rco) (IU/mL)
• Measurement method Platelet aggregation method
• the coagulation activity of blood coagulation factors in the plasma solution was measured using Sysmec's automatic coagulation measuring device CS2400 and the device's dedicated reagents.
• vWF antigen amount (vWF:Ag) (Measurement method: sandwich ELISA method) (Standard product) Standard human plasma for blood coagulation test (SIEMENS/Sysmex) (Immobilized antibody) Polyclonal Rabbit Anti-Human von Willebrand Factor antibody (abcam) (Detection antibody) Polyclonal Rabbit Anti-Human von Willebrand Factor antibody/HRP (Dako) (Test method) 25 ng/mL of Polyclonal Rabbit Anti-Human von Willebrand Factor antibody (abcam), which is a solid-phase antibody solution, was placed in a 96-well microplate at 100 ⁇ L/well and allowed to stand overnight at 4°C.
• Protein S activity (measurement method: clotting time method) The activity of free protein S in the sample was measured using the prothrombin time method. Protein S activity was measured by measuring the degree of prothrombin time prolongation using tissue factor, synthetic phospholipid, calcium chloride, and activated protein C as reagents. Since protein S activity correlates with prolongation of clotting time by adding protein S-deficient plasma to the diluted sample, the temporal change was measured to determine the protein S activity (%) in the sample.
• the blood coagulation factors that are easily deactivated by powdering were Fibrinogen and Factor VIII.
• Blood coagulation factors with less deactivation were Protein C, ⁇ 2-antiplasmin, Protein S, and VWF:Rco, and a common tendency was observed among the three nozzles.
• the dispenser nozzle showed the least deactivation of blood coagulation factors.
• blood coagulation factor activity when a dispenser nozzle was used, a negative correlation was observed between linear flow rate and activity recovery rate. It was found that the deactivation of can be suppressed.
• Example 3 Physicochemical properties and biological activity of dried plasma of the present invention [Summary] 1) Using 3 lots of SDP as a raw material, 6 lots of dried plasma were prepared using the freeze-vacuum dryer 1 (hereinafter, the prepared plasma will be referred to as "dried plasma"), and reproducibility was confirmed. 2) The lot-to-lot reproducibility of powder properties and quality was good, indicating that stable production of "dried plasma” was possible. 3) The particle size of the "dried plasma” was D50: 362.8 ⁇ 11.7 ⁇ m (6 lots), and the "dried plasma" prepared with a nozzle diameter of ⁇ 200 ⁇ m was about 160 ⁇ m larger than the nozzle diameter.
• the particle dispersibility was (D90-D10)/D50: 0.37 ⁇ 0.04, and the powder was homogeneous with high monodispersity.
• the moisture content was 3% or less.
• the content of coarse particles of 1000 ⁇ m or more was 6.3 ⁇ 5.3% (6 lots), and it was observed that the protein recovery rate tended to decrease when the coarse particle content was high.
• the reconstitution time for "dried plasma” was within 3 minutes for all lots. 6) Coarse particles had no effect on reconstitution time and clotting factor activity in the plasma solution after lysis.
• SDP used as a plasma raw material has a standard value of activity of blood coagulation factors FVIII, FV, FXI, Protein C, and ⁇ 2-antiplasmin of European Pharmacopoeia (EP) 10.0 “Human Plasma (Pooled and Treated F or Virus Inactivation ( 01/2020:1646)”.
• EP European Pharmacopoeia
• the activities of FVIII, FV, FXI, Protein C, and ⁇ 2-antiplasmin in the plasma solution after reconstitution of "dried plasma” all meet the EP standard values.
• Ta. 8 The DSC thermogram of the plasma solution after reconstitution of "dried plasma” had a similar profile to the thermograms of the FPP pool solution and SDP, and no denaturation due to powdering was observed.
• Example plasma The particle size distribution and particle dispersibility of the dried plasma prepared in each example (also referred to as “example plasma”) were measured in the same manner as in Example 2. The results are shown in Tables 8 and 9, respectively.
• the particle diameters (Mean ⁇ S.D.) of the example plasma prepared with a nozzle diameter of ⁇ 200 ⁇ m are: D10: 301.3 ⁇ 12.1 ( ⁇ m), D50: 362.8 ⁇ 11.7 ( ⁇ m), D90: 434.3 ⁇ 15.8 ( ⁇ m), and each particle was about 160 ⁇ m larger than the nozzle diameter.
• the dispersibility (Mean ⁇ S.D.) of the particles was (D90-D10)/D50: 0.37 ⁇ 0.04, and the powder was homogeneous with high monodispersity.
• Coarse particle content Table 10 shows the coarse particle content (% by weight) of 1000 ⁇ m or more in the plasma of each Example. The coarse particle content was evaluated in the same manner as in Example 2.
• the coarse particle content (% by weight) of 1000 ⁇ m or more in the six Example plasmas was 6.3% ⁇ 5.3%.
• the "standard value of fibrinogen in the above table” was based on the standard value of "Octaplas LG (registered trademark)" (Octapharma), an SD-treated plasma preparation.
• the blood coagulation factor activities of the plasma of each example all met the EP standard values.
• FIG. 3 shows a schematic diagram of Thrombin Generation Assay (TGA) using the CAT system. Evaluation of Example plasma was performed using reconstituted plasma.
• Thrombin blood coagulation factor IIa
• Thrombin is an enzyme that converts fibrinogen to fibrin in the intrinsic and extrinsic coagulation cascades.
• thrombin In addition to fibrinogen, thrombin also has the role of limiting and activating blood coagulation factors V, VIII, XI, and XIII, and the role of activating protein C. Furthermore, thrombin also activates platelets, leukocytes, and vascular endothelial cells, and is an important coagulation factor in the hemostasis mechanism of blood clots. In clinical practice, in order to understand the relationship between coagulation factors and bleeding during bleeding, activated partial thromboplastin time (APTT), which reflects endogenous coagulation factors, and prothrombin time (PT), which reflects extrinsic coagulation factors, are measured by clotting time.
• APTT activated partial thromboplastin time
• PT prothrombin time
• thrombin generation assay TGA method
• tissue factor (TF) forms a complex with blood coagulation factor VIIa
• blood coagulation factors IX and X are activated, resulting in a trace amount of Thrombin is generated.
• This thrombin causes blood coagulation factors VIII and V to activate platelets, and the activation of blood coagulation factors occurs on activated platelets, thereby increasing the thrombin generation reaction (also referred to as propagation).
• This platelet-dependent increase in thrombin production (also called thrombin burst) is necessary for stable fibrin formation that is resistant to fibrinolysis.
• thrombin burst can obtain data on the total amount of thrombin generated and the time of thrombin generation based on the thrombin concentration profile over time during thrombin generation, so it is also used to evaluate clinical conditions such as coagulopathy. .
• parameters for thrombin generation are Lag Time (time until thrombin generation starts (min)), ETP (total thrombin generation amount (nmol/L x min)), and Peak Thrombin ( The maximum thrombin generation amount (nmol/L)) and the Time to Peak (ttPeak) (time to the maximum thrombin generation value (min)) are determined.
• Lag Time corresponds to the initial phase of coagulation.
• Peak Thrombin reflects thrombin burst. Time to Peak reflects the pre-coagulation phase up to fibrin formation. Free thrombin, which converts fibrinogen to fibrin, is suppressed back to baseline after the time to reach maximum thrombin generation (ttPeak).
• the hemostatic effect can be estimated by evaluating these parameters. For example, when normal plasma is measured by the TGA method, Lag Time 2-4.33 (min), ETP 694.50-2625.67 (nmol/L ⁇ min), and Peak Thrombin 109.29-545.76 (nmol /L), ttPeak 3.89 to 8.50 (min) (Reference: OmidR. Zekavat. et al. Clinical and Applied Thrombosis/Hemostasis. 2013.00 (0) 1-8 ).
• the TGA method is a method of measuring thrombin produced by adding tissue factor (TF), calcium chloride, and phospholipid to citrated plasma using a synthetic substrate method.
• the measurement reagents are tissue factor reagent PPP Low (TF concentration 1 pmol/L), calcium chloride, and phospholipids. These reagents are added to the plasma sample, the fluorescence intensity is measured over time, and Lag is determined from the thrombogram.
• Time time until thrombin generation starts (min)
• ETP total thrombin amount (nmol/L x min)
• Peak Thrombin maximum thrombin generation amount (nmol/L)
• ttPeak Time to Peak
• Thrombinoscope Thrombin Calibrator
• PPP-Reagent Low Thrombinoscope
• Fluka-Reagent is prepared by adding 40 ⁇ L of Fluo-Substrate to 1 vial of Fluo-Buffer) (Measuring method) 20 ⁇ L of PPP-Reagent Low or Thrombin Calibrator and 80 ⁇ L of the measurement sample were placed in a 96-well microplate. It was set in a plate reader and incubated at 37 degrees for 10 minutes. 20 ⁇ L of FluCa Reagent was added to each well. Fluorescence intensity was measured for 60 minutes and a thrombogram was obtained.
• the dried plasma of the present invention has sufficient thrombin generation ability within the range of normal plasma without the risk of thromboembolism, and since it generates more thrombin than FFP, it is extremely useful as a hemostatic agent. That is clear.
• Example 4 Comparison of physical properties with dried plasma using powdered conventional technology
• the physical properties of the dried plasma of the present invention were compared with the physical properties of dried plasma prepared by Atmosphere Spray drying (ASD), which is a conventional powdering technique.
• ASD Atmosphere Spray drying
• Table 19 shows a summary of the physical property comparison results between Example 4 plasma and Comparative Example plasma.
• Example 3A1, 3A2, 3B1, 3B2, 3C1 and 3C2 Particles for six plasmas (Examples 3A1, 3A2, 3B1, 3B2, 3C1 and 3C2) prepared in Example 3 (the equipment parameters during preparation are the same as in Example 4), although the raw material lots are different.
• the particle diameter data and the particle diameter data for the plasma of Example 4 described above were aggregated, and the values of "average ⁇ S.D.” calculated based on the aggregated values are shown in Table 21 below.
• Coarse particle content Regarding the powder characteristics of dried plasma, it is important in manufacturing that it does not require a classification operation or is easy to classify, that the recovery rate of the powder is high, and that there are no problems such as nozzle clogging during dispensing. It is. Particularly, when coarse particles of 1000 ⁇ m or more, which are visible to the naked eye, are mixed into the preparation, the recovery rate of the powder decreases. A sieving test was conducted using a mesh (1000, 500, 300, 180, 100, 53 ⁇ m openings), and the weight of the remaining powder on the mesh after classification of dried plasma was measured to create a weight particle size distribution (see Table 22). ), the content of coarse particles of 1000 ⁇ m or more in plasma of Example 4 and plasma of comparative example was compared.
• Example 4 The content of coarse particles of 1000 ⁇ m or more was 0.1 (wt%) in the plasma of Example 4, while it was 32.3 (wt%) in the plasma of Comparative Example.
• Example 4 It was found that plasma had a particle size D50 of around 300 ⁇ m and had high monodispersity, so that coarse particles were unlikely to be formed.
• Figure 5 shows the weight particle size distribution of Example 4 plasma and Comparative Example plasma (in the figure, "Example 4 plasma” is written as MFD-PP. written).
• Dissolution time of dried plasma Dried plasma was dissolved at room temperature using a dedicated dissolution solution (20 mmol/L Glycine, pH 2.4), and the time until reconstitution was completed (dissolution time) was measured.
• the amount of lysis solution added was such that the protein concentration of the plasma solution after lysis was about 60 mg/mL (see Table 23).
• 5.0 mL of the solution was added to 0.5 g of dried plasma so that the weight ratio was 10 w/v%.
• Table 24 shows the dissolution time of each sample studied above.
• Example 4 plasma and the Comparative Example plasma were dissolved in about 5 minutes, but the Example 4 plasma was dissolved about 1 minute faster than the Comparative Example plasma. From the above, it is clear that the dried plasma of the present invention can be reconstituted into a solution state in a short period of time, and therefore has high practicality and usefulness in medical situations where emergencies such as blood transfusions are required.
• Example 4 Plasma / Comparative Example Plasma
• reconstituted plasma in the plasma solution (hereinafter also referred to as "reconstituted plasma” in this example) after reconstitution under the conditions described in "3) Dissolution time of dried plasma” above.
• the number of insoluble fine particles of ⁇ 100 ⁇ m was measured.
• Equipment Liquid particle counter HIAC System 9703+ (Beckman Courter)
• Sensor model HRLD150 Sampler settings: syringe size 1mL, probe size 2.75, 1/16O. D.
• the number of insoluble fine particles of 10 ⁇ m or more and 25 ⁇ m or more in the plasma solution after plasma reconstitution is significantly higher than that of dried plasma obtained by the conventional spray drying method. It is decreasing. Since insoluble fine particles can cause blood clots, it has been revealed that the dried plasma of the present invention is highly practical and has a very low risk of side effects after administration of reconstituted plasma to humans. .
• the "standard value of fibrinogen in the above table” was based on the standard value of the SD Plasma preparation Octaplas LG (registered trademark) (Octapharma). Each measurement value is MEAN ⁇ S. after three measurements. D. It is a value.
• Table 27 shows the activity recovery rate of each blood coagulation factor in dried plasma (MEAN ⁇ S.D. value of three measurements).
• the activity recovery rate is a ratio when the activity recovery value in FFP is taken as 100%.
• vWF has a multimer structure (multimer) in which a dimer structure forms a polymer with SS bonds, and is classified into three sizes: small multimers, medium multimers, and large-multimers.
• SDS-multimer analysis is generally used as a method for analyzing the higher-order structure of vWF, and analysis is performed using submarine electrophoresis and Western blotting. After vWF is separated by molecular weight size in a electrophoresis gel by electrophoresis, the molecular weight bands of vWF multimers are determined by Western blotting using an anti-vWF polyclonal IgG antibody as the primary antibody and a fluorescently labeled IgG antibody that binds to the primary antibody as the secondary antibody. Detect.
• the molecular weight size of vWF multimers is defined by the band position of the molecular weight, the 1st to 5th bands from the lowest molecular weight are small multimers, the 6th to 10th bands are medium multimers, and the 11th and higher bands are large-multimers. It is. It is known that vWF large-multimers, which have large multimer sizes, have a high primary clot-forming ability, and when vWF large multimers are cleaved and become low-molecular-weight, the platelet binding ability decreases and the hemostasis ability decreases.
• the activities of coagulation factors Factor V and Protein C which have EP standard values, met the EP standard values in all dried plasmas.
• the comparative plasma it was confirmed that many coagulation factors were inactivated, and in particular, high molecular weight proteins such as vWF and Fibrinogen were found to be significantly inactivated. No such decrease in activity was observed in the plasma of Example 4. Therefore, the dried plasma of the present invention has at least the following points: 1) It is powdered while maintaining the activities of vWF and Fibrinogen (high molecular weight protein), and 2) It is powdered while maintaining the vWF ristocetin activity. It has become clear that this method is superior to dried plasma obtained by spray-drying in that respect.
• Thrombin generation was measured in dried plasma, FFP, and SDP of Example 4 and Comparative Example, and each parameter was compared. The values were higher than those of FFP, SDP, and comparative plasma.
• FIG. 6 shows a diagram outlining the parameters of ROTEM measurement (Reference: Nobuyuki Katori. Journal of the Nichirin Asakai. 2013; 33, 263-271).
• ROTEM measurement parameters ⁇ CT (Clotting time: clotting time (sec)) Time from start of measurement to initial clot formation (until amplitude reaches 2 mm). Corresponds to APTT, PT, etc.
• ⁇ CFT Peak formation time: clot formation time (sec)
• ⁇ MCF Maximum clot firmness: Maximum clot firmness (mm)
• Maximum amplitude during measurement The larger the value, the stronger the blood clot.
• ⁇ ML Maximum solubility (%)
• Platelets in the blood and vWF bind together via GPIb- ⁇ /IX receptors on the platelet surface to form a primary thrombus (also referred to as a white thrombus), resulting in platelet adhesion.
• Fibrinogen binds to this primary thrombus via platelet membrane glycoprotein GPIIb/IIIa, resulting in platelet aggregation.
• the coagulation cascade is accelerated and the generated thrombin converts fibrinogen into fibrin, and the formed fibrin is crosslinked by Factor XIII to form a strong secondary thrombus (also called brown thrombus). formation, leading to hemostasis.
• vWF in the plasma was significantly inactivated, and it is thought that the ability to bind to platelets was insufficient, resulting in a decrease in the amount of primary thrombi.
• fibrinogen and Factor XIII were significantly inactivated, there was less fibrinogen as a substrate to be degraded by thrombin, and the low activity of Factor XIII resulted in insufficient crosslinking and only weak clots were formed. It was considered a thing.
• the present invention provides dried plasma useful in the pharmaceutical field, which has excellent particle homogeneity and maintains biological activity, and a method for producing the same.

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