WO2022202938A1 - 凍結乾燥用容器 - Google Patents

凍結乾燥用容器 Download PDF

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Publication number
WO2022202938A1
WO2022202938A1 PCT/JP2022/013715 JP2022013715W WO2022202938A1 WO 2022202938 A1 WO2022202938 A1 WO 2022202938A1 JP 2022013715 W JP2022013715 W JP 2022013715W WO 2022202938 A1 WO2022202938 A1 WO 2022202938A1
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WIPO (PCT)
Prior art keywords
freeze
liquid
dried
container
drying
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PCT/JP2022/013715
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English (en)
French (fr)
Japanese (ja)
Inventor
勇輔 望月
英生 伏見
伸彦 加藤
Original Assignee
富士フイルム株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by 富士フイルム株式会社 filed Critical 富士フイルム株式会社
Priority to CN202280024021.XA priority Critical patent/CN117043535A/zh
Priority to JP2023509268A priority patent/JPWO2022202938A1/ja
Priority to EP22775720.0A priority patent/EP4300022A4/en
Publication of WO2022202938A1 publication Critical patent/WO2022202938A1/ja
Priority to US18/474,051 priority patent/US20240053099A1/en

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    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F26—DRYING
    • F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
    • F26B25/00—Details of general application not covered by group F26B21/00 or F26B23/00
    • F26B25/06—Chambers, containers, or receptacles
    • F26B25/14—Chambers, containers, receptacles of simple construction
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/90—Preservation of foods or foodstuffs, in general by drying or kilning; Subsequent reconstitution
    • A23B2/92—Freeze drying
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F26—DRYING
    • F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
    • F26B25/00—Details of general application not covered by group F26B21/00 or F26B23/00
    • F26B25/06—Chambers, containers, or receptacles
    • F26B25/063—Movable containers or receptacles, e.g. carts, trolleys, pallet-boxes
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F26—DRYING
    • F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
    • F26B25/00—Details of general application not covered by group F26B21/00 or F26B23/00
    • F26B25/06—Chambers, containers, or receptacles
    • F26B25/14—Chambers, containers, receptacles of simple construction
    • F26B25/18—Chambers, containers, receptacles of simple construction mainly open, e.g. dish, tray, pan, rack
    • 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 disclosure relates to freeze-drying containers.
  • Freeze-drying containers are widely used for freeze-drying various liquids to be freeze-dried. Such freeze-drying containers are disclosed, for example, in Japanese Utility Model Laid-Open No. 62-101728.
  • the present disclosure has been made in view of such circumstances, and the problem to be solved by one embodiment of the present disclosure is to provide a freeze-drying container that can obtain a freeze-dried body with uniform pores. That is.
  • the present disclosure includes the following aspects. ⁇ 1> Including a container body containing a liquid to be freeze-dried, and a first heat transfer body in contact with the liquid to be freeze-dried and a cold heat source,
  • the container body is a non-heat conductor or a second heat conductor at least partially in contact with the surface of the liquid to be freeze-dried and the cold heat source,
  • a first shortest distance between an arbitrary point on the surface of the liquid to be freeze-dried and the first heat transfer body, and a second shortest distance in the liquid surface between the arbitrary point and the second heat transfer body is 20 mm or less, Freeze-drying container.
  • the ratio of the area of the portion of the first heat conductor and the second heat conductor in contact with the freeze-dried liquid to the volume of the freeze-dried liquid contained in the freeze-drying container at 23 ° C. is 200 mm 2 /
  • ⁇ 4> The freeze-drying container according to any one of ⁇ 1> to ⁇ 3>, wherein the second heat conductor has a thermal conductivity of 15 W/m ⁇ K or more at 0°C.
  • ⁇ 5> The freeze-drying container according to any one of ⁇ 1> to ⁇ 4>, wherein the first heat transfer member is arranged perpendicular to the bottom surface of the container body.
  • ⁇ 6> The freeze-drying container according to any one of ⁇ 1> to ⁇ 5>, wherein at least part of the container body and the first heat transfer member contacting the liquid to be freeze-dried has a hydrophobic membrane. .
  • ⁇ 7> The freeze-drying container according to ⁇ 6>, wherein the entire container body and the first heat transfer element have a hydrophobic film.
  • ⁇ 8> The freeze-drying container according to ⁇ 6> or ⁇ 7>, wherein the hydrophobic film has a thickness of 1 mm or less.
  • a freeze-drying container in which a freeze-dried product with uniform pores can be obtained.
  • FIG. 1 is a schematic perspective view showing an example of a freeze-drying container.
  • FIG. 2 is a schematic plan view showing an example of a freeze-drying container.
  • FIG. 3 is a schematic plan view showing an example of a freeze-drying container.
  • FIG. 4 is a schematic perspective view showing an example of a freeze-drying container.
  • FIG. 5 is a schematic plan view showing an example of a freeze-drying container.
  • FIG. 6 is a schematic cross-sectional view showing an example of a freeze-drying container.
  • FIG. 7 is a schematic cross-sectional view showing an example of how a freeze-drying liquid is stored in a freeze-drying container.
  • FIG. 1 is a schematic perspective view showing an example of a freeze-drying container.
  • FIG. 2 is a schematic plan view showing an example of a freeze-drying container.
  • FIG. 3 is a schematic plan view showing an example of a freeze-drying container.
  • FIG. 4 is a schematic
  • FIG. 8 is a schematic cross-sectional view showing an example of how a freeze-drying liquid is stored in a freeze-drying container.
  • 9 is an optical microscope image of a tissue section of the freeze-dried product of Example 1.
  • FIG. 10 is an optical microscope image of a sample of the freeze-dried body of Example 2.
  • FIG. 11 is an optical microscope image of a sample of the freeze-dried body of Example 3.
  • FIG. 12 is an optical microscope image of a sample of the freeze-dried body of Example 4.
  • FIG. 13 is an optical microscope image of a specimen of the freeze-dried body of Example 5.
  • FIG. 14 is an optical microscope image of a sample of the freeze-dried product of Example 6.
  • FIG. 15 is an optical microscope image of a sample of the freeze-dried product of Comparative Example 1.
  • freeze-drying container According to the present disclosure will be described below.
  • a numerical range indicated using “to” means a range including the numerical values before and after “to” as the minimum and maximum values, respectively.
  • upper or lower limits described in a certain numerical range may be replaced with upper or lower limits of other numerical ranges described step by step.
  • upper or lower limits described in a certain numerical range may be replaced with values shown in Examples.
  • a combination of two or more preferred aspects is a more preferred aspect.
  • the amount of each component means the total amount of the multiple types of substances unless otherwise specified when there are multiple types of substances corresponding to each component.
  • the term "process” includes not only an independent process, but also if the intended purpose of the process is achieved, even if it cannot be clearly distinguished from other processes. .
  • the amino acid sequences of polypeptides are referred to by single-letter (e.g., "G” for glycine residues) or three-letter (e.g., "Gly” for glycine residues) abbreviations well known in the art. It may be expressed using In the present disclosure, "%" for amino acid sequences of polypeptides is based on the number of amino acid (or imino acid) residues unless otherwise specified.
  • identity with respect to the amino acid sequences of two polypeptides to be contrasted refers to the value calculated by the following formula.
  • the comparison (alignment) of multiple polypeptides should be carried out according to a conventional method so as to maximize the number of identical amino acid residues.
  • Identity (%) ⁇ (number of identical amino acid residues) / (alignment length) ⁇ x 100
  • a freeze-drying container includes a container body that stores a freeze-drying liquid, and a first heat transfer body that is in contact with the freeze-drying liquid and a cold heat source.
  • the container body is a non-heat conductor or a second heat conductor at least partially in contact with the surface of the liquid to be freeze-dried and the cold heat source.
  • a first shortest distance between an arbitrary point on the liquid surface of the freeze-dried liquid and the first heat transfer body, and a second shortest distance in the liquid surface between the arbitrary point and the second heat transfer body At least one of the shortest distances is 20 mm or less.
  • the freeze-dried material obtained by freeze-drying the liquid to be freeze-dried has a pore structure, and the pores are contained in the freeze-dried material as air bubbles.
  • a freeze-drying container a container provided with a partition plate as described in Japanese Utility Model Laid-Open No. 62-101728 is known.
  • simply using a freeze-drying container equipped with a partition plate tends to make the size and state of pores formed in the dried body after the freeze-dried body uneven, and the freeze-dried body with uniform pores can be difficult to obtain.
  • the first shortest distance from the first heat transfer body and the distance from the second heat transfer body to any point on the surface of the liquid to be freeze-dried At least one of the in-plane second shortest distances is 20 mm or less. That is, since any point on the surface of the liquid to be freeze-dried can be within 20 mm from at least one of the first heat transfer body and the second heat transfer body, a freeze-dried body having uniform pores can be obtained. Obtainable.
  • the first heat transfer body and the second heat transfer body are sometimes simply referred to as "heat transfer bodies”.
  • heat transfer material refers to a material with a thermal conductivity of 5 W/m ⁇ K or higher at 0°C. Thermal conductivity is a value measured by a laser flash method. Objects other than heat conductors are defined as "non-heat conductors”.
  • the shape of the first heat conductor is not particularly limited, and may be, for example, plate-like or rod-like.
  • the position of the first heat conductor provided inside the container body is not particularly limited.
  • the first heat transfer body in contact with the freeze-dried liquid and the cold heat source may be arranged perpendicular to the bottom surface of the container body. Accordingly, when the cold heat source is arranged in contact with the bottom of the container body, the first heat transfer body can be efficiently cooled through the bottom.
  • perpendicular means 90° ⁇ 15°.
  • the material of the first heat conductor is not particularly limited as long as the thermal conductivity at 0° C. is 5 W/m ⁇ K or more, and is preferably made of metal, for example.
  • the metal is preferably aluminum, gold, silver, copper, iron, alloys thereof (eg, aluminum alloy (eg, A5052), stainless steel, etc.), and more preferably aluminum or its alloys.
  • the thermal conductivity of the first heat transfer body at 0° C. is preferably 15 W/m K or more, more preferably 50 W/m K or more. is more preferable, more preferably 100 W/m ⁇ K or more, and even more preferably 150 W/m ⁇ K or more.
  • the number of first heat conductors may be one, or two or more.
  • the first heat conductors may be independent of each other, and at least some of them may be connected.
  • the mode of connection is not particularly limited.
  • the thermal conductivity between the heat conductors is preferably 15 W/m ⁇ K or more, more preferably 50 W/m ⁇ K or more, and even more preferably 100 W/m ⁇ K or more.
  • at least a part of the first heat transfer body and the second heat transfer body may be connected.
  • the mode of connection is not particularly limited, it is preferable to be thermally connected, and the thermal conductivity between the first heat transfer body and the second heat transfer body that are connected is 15 W/m ⁇ K or more. , more preferably 50 W/m ⁇ K or more, and even more preferably 100 W/m ⁇ K or more.
  • the first heat transfer body may function as a partition member.
  • the freeze-drying container may be provided with a plurality of storage portions which are not in communication with each other and which contain the liquid to be freeze-dried.
  • the plurality of storage units may store the same freeze-dried liquid or may store different freeze-dried liquids.
  • the plurality of storage units may store the same volume of the freeze-dried liquid, or may store different volumes of the freeze-dried liquid.
  • the container body is a container that contains the liquid to be freeze-dried.
  • the container body is a non-heat conductor or a second heat conductor at least partially in contact with the surface of the liquid to be freeze-dried and the cold heat source.
  • the shape of the container body is not particularly limited, and may be, for example, a rectangular parallelepiped shape, a cylindrical shape, or the like.
  • the material of the container body is not particularly limited, and examples thereof include a non-heat conductor such as resin and a second heat conductor such as metal.
  • the size of the container body is not particularly limited, and even if the container body is large, a freeze-dried product with uniform pores can be obtained.
  • a freeze-drying container in which the container body is formed of a heat conductor but no heat conductor is arranged in the container body, uniform
  • the accommodating portion of the container body is narrow in plan view, thereby reducing the distance from an arbitrary point of the freeze-dried liquid to the container body (heat transfer body). Therefore, uniform freeze-drying can be easily performed.
  • the maximum length of the contour of the housing portion of the container body (that is, the maximum length of the straight line connecting any two points of the contour) is about less than 20 mm in plan view.
  • the maximum length of the contour is, for example, the diagonal if the housing portion of the container body is square, the diameter if it is circular, or the major axis if it is elliptical.
  • the heat transfer body is arranged in the container body, and the liquid to be freeze-dried By setting the shortest distance between the two to 20 mm or less, uniform freeze-drying can be performed, and a freeze-dried product having uniform pores can be obtained.
  • the maximum length of the contour of the housing portion of the container body may be, for example, 30 mm or longer, 50 mm or longer, 90 mm or longer, or longer in plan view.
  • the freeze-drying container according to the present disclosure is effective when freeze-drying a large amount of liquid to be freeze-dried.
  • a certain length for example, 5 cm
  • the volume of the liquid to be freeze-dried increases, the dry state of the liquid to be freeze-dried tends to differ between the top and bottom of the specific container, and a freeze-dried body with uniform pores can be obtained. can be difficult.
  • the size of the container body can be appropriately selected according to the amount of the freeze-dried liquid to be freeze-dried, and the heat transfer body can be appropriately provided. can.
  • the volume of the liquid to be freeze-dried can be kept within an appropriate range, making it easy to obtain a freeze-dried product having uniform pores.
  • the entire container body may be one second heat transfer body.
  • the freeze-drying container may be, for example, one in which one or more first heat conductors are connected within a container body composed of a second heat conductor.
  • the freeze-drying container may be a single (that is, integral) heat transfer body as a whole.
  • the freeze-drying container may have, for example, a first heat transfer body that is integral with the container body and arranged inside the container body that is composed of the second heat transfer body.
  • the freeze-drying container of such an aspect can be manufactured, for example, by cutting a metal so that the second heat transfer body and the first heat transfer body, which are the main body of the container, are integrated.
  • the freeze-drying container 100 shown in FIGS. 1 and 2 includes a rectangular parallelepiped container body 10 and two plate-shaped first heat conductors 30 perpendicular to the bottom surface of the container body 10,
  • the first heat transfer body 30 functions as a partition plate and is divided into three storage sections 50 that store the liquid to be freeze-dried.
  • the heat body 30 functions as a partition plate and is divided into five storage sections 50 that store the liquid to be freeze-dried.
  • the freeze-drying container 300 shown in FIGS. 4 and 5 includes a rectangular parallelepiped container body 10 and a rod-shaped first heat conductor 32 perpendicular to the bottom surface of the container body 10 .
  • the shape of the longitudinal cross section may be a rounded shape or an inclined tapered shape.
  • the freeze-drying container has an arbitrary point on the liquid surface of the liquid to be freeze-dried and the first shortest distance (hereinafter sometimes simply referred to as "first shortest distance”), and the arbitrary point and the second heat transfer At least one of the second shortest distance in the liquid surface to the body (hereinafter sometimes simply referred to as "second shortest distance”) is 20 mm or less.
  • first shortest distance is 20 mm or less.
  • a first shortest distance between an arbitrary point P1 on the surface and a point P2 on the first heat conductor 30 is 20 mm or less.
  • the container body 10 is the second heat transfer body, for example, the first shortest distance between the points P1 and P2, and the point P1 and the second heat transfer body (one part of the container body 10) Part
  • At least one of the second shortest distances in the liquid surface between the above point P3 is 20 mm or less.
  • a first shortest distance between an arbitrary point P4 on the surface and a point P5 on the first heat conductor 30 in the freeze-dried liquid 70 is 20 mm or less.
  • the first shortest distance between the point P4 and the point P5 and the second heat transfer body (one part of the container body 10) from the point P4 Part) At least one of the second shortest distances in the liquid surface between the upper point P6 is 20 mm or less.
  • the first shortest distance is preferably 15 mm or less. Moreover, from the viewpoint of making the pores more uniform, the first shortest distance is preferably 5 mm or more. Similarly, from the viewpoint of obtaining a freeze-dried product having uniform pores in a short time, the second shortest distance is preferably 15 mm or less. Moreover, from the viewpoint of making the pores more uniform, the second shortest distance is preferably 5 mm or more.
  • the material of the second heat conductor is not particularly limited as long as the thermal conductivity at 0° C. is 5 W/m ⁇ K or more, and is preferably made of metal, for example.
  • the metal is preferably aluminum, gold, silver, copper, iron, alloys thereof (eg, aluminum alloy (eg, A5052), stainless steel, etc.), and more preferably aluminum or its alloys.
  • the thermal conductivity of the second heat transfer body at 0° C. is preferably 15 W/m K or more, more preferably 50 W/m K or more. is more preferable, more preferably 100 W/m ⁇ K or more, and even more preferably 150 W/m ⁇ K or more.
  • the thermal expansion coefficient of the container body material is preferably 10 ⁇ 10 ⁇ 5 /K or less, more preferably 50 ⁇ 10 ⁇ 6 /K or less, and 25 ⁇ It is more preferably 10 ⁇ 6 /K or less.
  • the material of the container body is preferably metal, for example.
  • the first heat transfer element and the second heat transfer element (when the container body does not contain the second heat transfer element, the first heat transfer
  • the ratio of the area of the portion of the body) in contact with the liquid to be freeze-dried (hereinafter sometimes referred to as "the ratio of the liquid contact area to the liquid volume”) is preferably 200 mm 2 /mL or more.
  • the contact area of the freeze-dried liquid with respect to the heat conductor per unit volume can be increased. Therefore, it becomes easier to obtain a freeze-dried product having uniform pores in a short period of time. More preferably, the ratio of the wetted area to the liquid volume is 210 mm 2 /mL or more.
  • the ratio of the liquid contact area to the liquid volume is preferably 1500 mm 2 /mL or less, more preferably 1000 mm 2 /mL or less, and even more preferably 500 mm 2 /mL or less.
  • the contact area with the first heat conductor and the second heat conductor (the first heat conductor when the container body does not contain the second heat conductor) per unit volume of the liquid to be freeze-dried is Since it can be increased, the volume of the freeze-dried liquid per unit contact area of the heat transfer body can be increased, that is, the height (bulk) of the freeze-dried liquid can be increased.
  • the ratio of the liquid contact area to the liquid volume satisfies the above range means that at least one container has a liquid contact area and It means that the ratio to the liquid volume satisfies the above range.
  • the container body, the first heat conductor, and the second heat conductor are arranged so that the ratio of the liquid contact area to the liquid volume is within the above range.
  • the shape, size, etc. may be selected as appropriate.
  • the freeze-drying container includes at least one part of the container body (which may include the second heat transfer body) and the first heat transfer body that contacts the liquid to be freeze-dried.
  • a hydrophobic film may be attached to the portion, or a hydrophobic film may be attached to the entire container body (which may include the second heat transfer body) and the first heat transfer body.
  • the material of the hydrophobic membrane is not particularly limited, and includes fluorine-based materials, fluorine-based resins, silicone resins, aromatic polymers, polyethylene-based resins, polypropylene-based resins, polystyrene-based resins, and the like.
  • the hydrophobic membrane preferably contains a fluorine-based material or an aromatic polymer, and particularly preferably a fluorine-based material.
  • fluorine-based materials examples include FEP (tetrafluoroethylene-hexafluoropropylene copolymer (also called perfluoroethylene propene copolymer)), fluorine resin, and nickel eutectoid material.
  • the hydrophobic membrane may be formed by subjecting a desired portion of the freeze-drying container to a hydrophobic treatment.
  • the method of applying the hydrophobic treatment is not particularly limited, and examples thereof include solution coating, plating, vapor deposition, and the like.
  • Hydrophobic plating may be applied to the freeze-drying vessel or parts thereof by known methods. Further, for example, there is a method of forming a eutectoid film of fluororesin and nickel by using “Nif Grip” (registered trademark) manufactured by ULVAC TECHNO CO., LTD. or “Caniflon” (registered trademark) manufactured by Nippon Kanigen Co., Ltd. Further, for example, there is a method of forming an aromatic polymer film by vapor deposition of Parylene manufactured by Japan Parylene LLC.
  • the thickness of the hydrophobic membrane is not particularly limited. From the viewpoint of thermal conductivity between the heat transfer body and the liquid to be freeze-dried, the thickness of the hydrophobic membrane is preferably 1 mm or less, more preferably 500 ⁇ m or less, and further preferably 250 ⁇ m or less. preferable. The lower limit of the thickness of the hydrophobic membrane may be about 0.5 ⁇ m.
  • the freeze-drying container may contain other members than the above.
  • the freeze-drying container may contain, for example, a partition member other than the heat conductor.
  • Cold heat source is in contact with a heat transfer body that is in contact with the freeze-dried liquid, and freezes the freeze-dried liquid via the heat transfer body.
  • the cold heat source is not particularly limited as long as it can freeze the liquid to be freeze-dried, and may include, for example, solids, liquids, supercritical fluids, etc. of 0°C or lower as refrigerants.
  • the form of the cold heat source is not particularly limited, and for example, it is preferably a metal plate in which a liquid coolant is circulated.
  • the number of cold heat sources in contact with the freeze-drying container is not particularly limited, and may be one or two or more. It may be appropriately selected in consideration of the size of the heat transfer bodies, the presence or absence of thermal connection between the heat transfer bodies, and the like. For example, in the case of a freeze-drying container in which the container body is the second heat conductor and the first heat conductor and the second heat conductor are integrated, the first heat conductor and the second heat conductor are Because of the overall thermal connection, freeze-drying may be performed with a single cold source located at the bottom of the freeze-drying container.
  • the liquid to be freeze-dried is not particularly limited, and examples thereof include biocompatible polymers such as gelatin and collagen, or aqueous solutions thereof.
  • the polymer porous body is preferably a porous block made of a polymer.
  • the porous polymer body is a frozen polymer aqueous solution obtained by placing a polymer-containing aqueous polymer solution (liquid to be freeze-dried) in a freeze-drying container, subjecting it to a freezing process, and freezing the aqueous polymer solution. After obtaining, the frozen polymer aqueous solution is subjected to a water removal step to remove the water.
  • the porous polymer body may be subjected to a pulverization process, a classification process, a cross-linking process, and the like, and is suitable as a material for cell scaffolds, transplant members, tissue repair materials, and the like.
  • a macromolecule refers to a molecule with a high molecular weight that has a structure composed of multiple repetitions of units derived substantially or conceptually from a molecule with a low molecular weight.
  • examples include polyamines, cellulose, amylose, starch, chitin, polypeptides, proteins, DNA and RNA.
  • the macromolecules are water soluble, more preferably polypeptides and proteins.
  • collagen and gelatin are particularly preferred.
  • the ratio of hydrophilic repeating units in the polymer is preferably 50% or less, more preferably 30% or less. If the hydrophilic unit ratio is higher than this, the free water around the polymer is reduced and freezing is inhibited.
  • the ratio of hydrophilic repeating units refers to the ratio of repeating units having ionic groups and/or hydroxyl groups in the polymer.
  • the aforementioned gelatin means a polypeptide containing 6 or more consecutive sequences represented by Gly-XY, and having 1 or more amino acid residues other than the sequence represented by Gly-XY in the polypeptide. You may have The sequence represented by Gly-XY is a sequence corresponding to an amino acid sequence derived from a partial amino acid sequence of collagen, and repetition of this sequence means a sequence characteristic of collagen.
  • a plurality of Gly-XY may be the same or different.
  • X and Y in the Gly-XY sequence are independent for each repeating unit and may be the same or different.
  • Gly-XY Gly represents a glycine residue
  • X and Y represent arbitrary amino acid residues other than glycine residues.
  • X and Y preferably contain many imino acid residues, ie, proline residues or oxyproline residues. The content of such imino acid residues preferably accounts for 10% to 45% of the gelatin as a whole.
  • the content of Gly-XY in the gelatin is preferably 80% or more, more preferably 95% or more, and most preferably 99% or more.
  • the above gelatin may be either a natural type or a mutant type that differs from the natural type in at least one amino acid residue.
  • Natural gelatin means gelatin made from naturally occurring collagen, or a polypeptide having the same amino acid sequence as gelatin made from naturally occurring collagen. Unless otherwise specified, mutant or recombinant gelatins are collectively referred to as recombinant gelatins in this disclosure.
  • Examples of natural gelatin or recombinant gelatin thereof include those derived from animals such as fish and mammals, and natural gelatin of mammals or recombinant gelatin thereof is preferable. Examples of mammalian animals include humans, horses, pigs, mice, rats and the like, with humans or pigs being more preferred.
  • the natural gelatin is preferably derived from porcine or human, and the recombinant gelatin is preferably human-derived recombinant gelatin.
  • one or more bases or amino acid residues are added to the base sequence or amino acid sequence of the collagen-encoding gene having 6 or more consecutive sequences represented by Gly-XY.
  • Recombinant gelatin obtained by introducing a modified base sequence or amino acid sequence into an appropriate host and expressing it by a conventional method is preferred.
  • the (bone) tissue repair ability can be enhanced, and various properties can be expressed as compared with the case of using natural gelatin. It has the advantage of being able to avoid adverse effects.
  • the recombinant gelatin examples include, for example, European Publication No. 1014176, US Pat. Those disclosed in JP-A-2010-518833, JP-A-2010-519251, WO 2010/128672 and WO 2010/147109 can be particularly preferably used.
  • the recombinant gelatin preferably has a molecular weight of 2 kDa or more and 100 kDa or less, more preferably 5 kDa or more and 90 kDa or less, and more preferably 10 kDa or more and 90 kDa or less.
  • the recombinant gelatin preferably further contains a cell adhesion signal, and the recombinant gelatin preferably has two or more cell adhesion signals per molecule. , more preferred.
  • cell adhesion signals include the RGD, LDV, REDV, YIGSR, PDSGR, RYVVLPR, LGTIPG, RNIAEIIKDI, IKVAV, LRE, DGEA, and HAV sequences.
  • RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence and HAV sequence are preferred, and RGD sequence is particularly preferred.
  • the ERGD sequence is more preferred.
  • the number of amino acid residues between RGDs is preferably 0-100, more preferably 25-60. Moreover, it is preferable that the RGD sequences are arranged non-uniformly within such a range of amino acid residue numbers.
  • the ratio of the RGD sequence to the total number of amino acid residues in the recombinant gelatin is preferably at least 0.4%, and when the recombinant gelatin contains 350 or more amino acid residues, each Preferably the stretch contains at least one RGD sequence.
  • the recombinant gelatin preferably contains at least 2 RGD sequences, more preferably at least 3 RGD sequences, and even more preferably at least 4 RGD sequences per 250 amino acid residues.
  • the sequence of the recombinant gelatin preferably has the following aspects: (1) does not contain serine residues and threonine residues, (2) serine residues, threonine residues, asparagine residues, and tyrosine residues. (3) does not contain the amino acid sequence represented by Asp-Arg-Gly-Asp.
  • the above-mentioned recombinant gelatin may have aspects (1) to (3) of this preferred sequence alone, or may have two or more aspects in combination. Also, the recombinant gelatin may be partially hydrolyzed.
  • the recombinant gelatin preferably has a repeating structure of A-[(Gly-XY)n]mB.
  • m represents 2 to 10, preferably 3 to 5.
  • a and B represent any amino acid or amino acid sequence.
  • n represents 3 to 100, preferably 15 to 70, more preferably 50 to 60.
  • the recombinant gelatin has the formula: Gly-Ala-Pro-[(Gly-X-Y)63]3-Gly (wherein each of the 63 Xs independently represents any amino acid residue, Each of the 63 Ys independently represents an amino acid residue, and the three (Gly-X-Y)63s may be the same or different.).
  • a plurality of naturally occurring collagen sequence units are bound to the recombinant gelatin repeating unit.
  • the naturally occurring collagens referred to herein preferably include types I, II, III, IV and V. More preferably, it can be type I, type II or type III.
  • Collagen is preferably derived from humans, horses, pigs, mice, and rats, with human beings being more preferred.
  • the isoelectric point of the recombinant gelatin is preferably 5-10, more preferably 6-10, still more preferably 7-9.5.
  • Preferred embodiments of the recombinant gelatin include the following: (1) carbamoyl groups are not hydrolyzed, (2) no procollagen, (3) no telopeptides. (4) Substantially pure collagen materials prepared with nucleic acids encoding native collagen.
  • the above-mentioned recombinant gelatin may have these preferred aspects (1) to (4) alone, or may have two or more aspects in combination.
  • the above recombinant gelatin is preferably any one of the following (A) to (C) because of its high ability to repair (bone) tissue.
  • A a polypeptide represented by SEQ ID NO: 1 below; GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP) 3G (SEQ ID NO: 1)
  • B having a partial sequence having 80% or more sequence identity with the partial amino acid sequence consisting of the 4th to 192nd amino acid residues in the amino acid sequence of (A), and (bone) tissue repair ability a polypeptide having
  • C A polypeptide comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted or added
  • sequence identity in (B) above is more preferably 90% or more, more preferably 95% or more, from the viewpoint of the (bone) tissue repair ability of the recombinant gelatin.
  • the partial amino acid sequence in the sequence (B) above is a partial amino acid sequence corresponding to the repeating unit of the sequence shown in SEQ ID NO:1.
  • the polypeptide should contain one, preferably two or more repeating units with sequence identity of 80% or more. be able to.
  • polypeptide defined in (B) above is a partial sequence having a sequence identity of 80% or more with the partial amino acid sequence corresponding to the repeating unit, as the total number of amino acid residues, preferably contains 80% or more of
  • the length of the polypeptide defined in (B) above can be 151 to 2260 amino acid residues, and from the viewpoint of post-crosslinking degradation, 193 or more, and from the viewpoint of stability. , preferably 944 or less amino acid residues, more preferably 380 to 756 amino acid residues.
  • polypeptide defined in (C) above consists of an amino acid sequence in which one or several amino acid residues are deleted, substituted or added with respect to the amino acid sequence (A) above, and has tissue repair ability.
  • the number of amino acid residues to be deleted, substituted, or added in the polypeptide defined in (C) above may be one or several, and varies depending on the total number of amino acid residues of the recombinant gelatin. , 2 to 15, preferably 2 to 5.
  • the recombinant gelatin can be produced by genetic recombination techniques known to those skilled in the art, for example, EP 1014176, US Pat. It can be produced according to the method described in . Specifically, a gene encoding a given recombinant gelatin amino acid sequence is obtained, incorporated into an expression vector to prepare a recombinant expression vector, and introduced into an appropriate host to prepare a transformant. do. Recombinant gelatin is produced by culturing the resulting transformant in an appropriate medium, and the recombinant gelatin used in the present disclosure is prepared by recovering the recombinant gelatin produced from the culture. be able to.
  • the aqueous polymer solution contains one or more polymers.
  • the polymer concentration in the aqueous polymer solution is preferably 0.1% by mass or more, more preferably 1% by mass or more, and particularly preferably 5% by mass or more. If the concentration is lower than 0.1% by mass, it is difficult to maintain the structure of the porous polymer after removing water. It is preferable that the aqueous polymer solution gels at a freezing temperature or higher.
  • the upper limit of the polymer concentration in the aqueous polymer solution is not particularly limited as long as the polymer can be dissolved, but is generally 40% by mass or less, and may be 30% by mass or less, or 20% by mass or less.
  • An aqueous polymer solution is prepared by purifying and concentrating a solution containing a polymer, or by dissolving a dry polymer in an aqueous medium. (1) It may be adjusted for use, or (2) it may be prepared and used in advance. (3) An aqueous polymer solution may be prepared by freeze-drying the aqueous polymer solution obtained by purification and concentration, and adding an aqueous medium to the resulting freeze-dried product to redissolve it. Alternatively, (4) the aqueous polymer solution obtained by purification and concentration may be frozen, and the obtained frozen body may be thawed to prepare the aqueous polymer solution. The frozen body is preferably thawed at 30 to 40° C.
  • the above method (4) is preferable from the viewpoints of reducing the time and effort required for preparation, convenience of transportation and storage, and reducing pores and insoluble matter in the aqueous polymer solution.
  • the pores and insoluble matter dispersed in the aqueous polymer solution are preferably removed before the freezing step by operations such as filtration, centrifugation, pressure reduction, and defoaming. As a result, the yield of the frozen macromolecule solution with low anisotropy is improved. Removal of pores and insoluble matter can be evaluated by turbidity measurement. Alternatively, it can be evaluated by visual inspection with an optical microscope. For example, the number of pores and undesired substances reflected in the field of view of an optical microscope can be calculated, and the number of pores and insoluble substances in 1 ⁇ L of the aqueous polymer solution can be evaluated.
  • the number of pores and insoluble matter in the aqueous polymer solution is preferably 0.5/ ⁇ L or less, more preferably 0.3/ ⁇ L or less, and further preferably 0.1/ ⁇ L or less. 0 cells/ ⁇ L is particularly preferable.
  • Components other than the polymer may be added to the aqueous polymer solution for the purpose of adding predetermined properties.
  • Such other ingredients include, for example, ingredients related to bone regeneration or osteogenesis, such as osteoinductive agents.
  • osteoinductive drugs include BMP (bone morphogenetic factor) and bFGF (basic fibroblast growth factor), but are not particularly limited.
  • Other examples include polypeptide or protein cross-linking agents.
  • the aqueous medium for the aqueous polymer solution is not particularly limited as long as it can dissolve the polymer and can be used for living tissue. Examples include water, physiological saline, phosphate buffer and the like. can be listed.
  • the gelatin concentration in the gelatin solution is not particularly limited as long as it is a concentration at which gelatin can be dissolved.
  • the gelatin concentration in the gelatin solution is, for example, preferably 0.5% by mass to 20% by mass, more preferably 2% by mass to 16% by mass, and 4% by mass to 12% by mass. More preferred.
  • the gelatin solution may be defoamed before the freezing step. This makes it easier to uniformly form ice crystals.
  • the defoaming method is not particularly limited, but for example, vacuum centrifugal defoaming can be performed at a pressure of 2 to 10 kPa.
  • the gelatin solution may be filtered to remove undissolved particles.
  • the filtering method is not particularly limited, for example, pressure filtering is performed using a filter with a pore size of 0.22 to 0.45 ⁇ m.
  • the material of the filter there are no particular restrictions on the material of the filter, and polytetrafluoroethylene, polyethersulfone, cellulose acetate, polyvinylidene fluoride, etc. can be used. is preferred.
  • the temperature at which the gelatin solution is prepared is not particularly limited, and may be a commonly used temperature, for example, 0°C to 60°C, preferably 3°C to 40°C.
  • freeze-drying process is a combination of the following freezing process and subsequent water removal process (drying process).
  • a step of freezing the aqueous polymer solution (liquid to be freeze-dried) contained in the freeze-drying container (hereinafter sometimes referred to as a “freezing step”) is performed. Thereby, a frozen polymer aqueous solution can be obtained.
  • freezing may be performed using a device such as a refrigerator or a freeze dryer. In the case of freezing with a freeze dryer, water can be continuously removed (freeze-dried) from the frozen polymer aqueous solution using the same apparatus.
  • the temperature in the freezing process varies depending on the type of polymer and the concentration of the aqueous polymer solution.
  • the temperature difference immediately before heat generation is preferably 2.5°C or less.
  • the "temperature difference immediately before the generation of heat of solidification” means the maximum value of the temperature difference between the portion with the highest liquid temperature and the portion with the lowest liquid temperature between 1 second and 10 seconds before the generation of heat of solidification. .
  • the temperature of the portion with the lowest liquid temperature is preferably ⁇ 5° C. or less, more preferably ⁇ 7.5° C. or less, and further preferably ⁇ 10° C. or less. preferable.
  • the temperature of the portion where the liquid temperature is the lowest is preferably -15° C. or higher.
  • the temperature of the portion with the lowest liquid temperature is most preferably -15°C to -7.5°C.
  • the temperature difference immediately before the heat of solidification is generated can be measured as follows. By inserting thermocouple thermometers at multiple points in the liquid to be freeze-dried and freeze-drying while recording the temperature over time with a data logger, the temperature immediately before the heat of solidification is generated can be measured. At that time, by measuring at least the temperature of the part furthest from the first heat conductor and the second heat conductor and the temperature of the part closest to the first heat conductor and the second heat conductor, " It is possible to measure the temperature difference just before the heat of solidification is generated.
  • the “anisotropy” of the frozen polymer aqueous solution means physical properties measured as follows. After freeze-drying the frozen aqueous polymer solution, the freeze-dried body (porous polymer body) is horizontally and vertically cut in the vicinity of its center. Each section is then stained and a defined area (2.0 mm x 2.0 mm or 2.5 mm x 2.5 mm) is observed under an optical microscope. Among the rectangles circumscribing the area surrounded by the dyed material in the observation area, the circumscribing rectangle having the maximum distance between two opposite sides of the rectangle is selected.
  • the length of the long side of the circumscribing rectangle where the distance between the two opposing sides is the maximum is measured for 50 pieces within the observation area in each of the horizontal cross section and the vertical cross section, and the average is the frozen body. It is the average value of the major diameter of the mesh. For each mesh at this time, the smaller one of the major diameter (average value) of the horizontal cross section and the major diameter (average value) of the vertical cross section is d1, and the other is d2. direction”.
  • the anisotropy of 3 or less is defined as "low anisotropy".
  • the yield of the frozen polymer aqueous solution with low anisotropy be 90% or more.
  • a step of removing water from the frozen aqueous polymer solution (hereinafter sometimes referred to as a "moisture removing step") is carried out. Thereby, a polymer porous body (freeze-dried body) can be obtained.
  • the means for removing water is not particularly limited, and includes a method of melting ice in the frozen polymer aqueous solution, a method of sublimation (freeze-drying), etc. Freeze-drying is preferred.
  • the freeze-drying period can be, for example, 0.5 hours to 300 hours. There are no particular restrictions on the freeze dryer that can be used.
  • Example 1 [Freeze-drying container] Using an aluminum alloy (A5052) with external dimensions of 100 mm (vertical) ⁇ 106 mm (horizontal) ⁇ 31 mm (height), the container body 10 with internal dimensions of 90 mm (vertical) ⁇ 96 mm (horizontal) ⁇ 28 mm (depth) (second heat conductor, wall thickness: 5 mm, bottom thickness: 3 mm) and two plate-shaped first heat conductors 30 (90 mm (vertical) ⁇ 3 mm (thickness) ⁇ 23 mm (height)) was made.
  • A5052 aluminum alloy
  • the container body 10 with internal dimensions of 90 mm (vertical) ⁇ 96 mm (horizontal) ⁇ 28 mm (depth) (second heat conductor, wall thickness: 5 mm, bottom thickness: 3 mm) and two plate-shaped first heat conductors 30 (90 mm (vertical) ⁇ 3 mm (thickness) ⁇ 23 mm (height) was made.
  • the walls of the container main body 10 and the first heat conductors 30 are arranged at regular intervals (30 mm) in the horizontal direction, and the freeze-drying container 100 is provided with three storage units 50 that are not in communication with each other. Furthermore, the container body 10 was integrated with the first heat transfer body 30 . In addition, the entire freeze-drying container 100 was subjected to hydrophobic plating (eutectoid film of fluororesin and nickel, thickness: 10 ⁇ m) using “Nif Grip” (registered trademark) manufactured by ULVAC TECHNO.
  • aqueous gelatin solution As a liquid to be freeze-dried, an aqueous gelatin solution was prepared in the following manner.
  • CBE3 As a recombinant gelatin, the following recombinant peptide CBE3 (described in International Publication No. 2008/103041) was prepared.
  • the amino acid sequence of CBE3 does not contain serine, threonine, asparagine, tyrosine and cysteine residues.
  • CBE3 has an ERGD sequence. Isoelectric point: 9.34 The proportion of hydrophilic repeating units in the polymer is 26.1%.
  • Amino acid sequence SEQ ID NO: 1
  • the gelatin aqueous solution was sampled in a polystyrene transparent container so as to have a liquid thickness of 2.5 mm, and was observed with an optical microscope in a field of view of 2.5 mm ⁇ 2.5 mm from the bottom surface to the top surface of the liquid at intervals of 100 ⁇ m. Observation of 10 fields of view was performed, and the average number of pores and insoluble matter was calculated.
  • Freeze-drying process 20.5 g of an aqueous gelatin solution was poured into each of the three containers of the freeze-drying container.
  • the volume of 20.5 g of the gelatin aqueous solution at 23° C. was 20.1 mL, and was measured by placing the gelatin aqueous solution in a graduated cylinder and comparing the mass and volume.
  • Place the freeze-drying container in a freeze dryer (manufactured by ULVAC, DFR-5N-B), allow to stand at 7°C for 2 hours, cool to -10°C, then -30°C for 14 hours, then -10. C. for 44 hours and then vacuum drying at 20.degree.
  • Example 2 [Freeze-drying container] Using an aluminum alloy (A5052) with external dimensions of 100 mm (vertical) ⁇ 106 mm (horizontal) ⁇ 31 mm (height), the container body 10 with internal dimensions of 90 mm (vertical) ⁇ 96 mm (horizontal) ⁇ 28 mm (depth) (second heat conductor, wall thickness: 5 mm, bottom thickness: 3 mm) and four plate-shaped first heat conductors 30 (90 mm (vertical) ⁇ 3 mm (thickness) ⁇ A lyophilization container 200 as shown in FIG.
  • a lyophilization container 200 as shown in FIG.
  • the walls of the container body 10 and the first heat conductors 30 are arranged at equal intervals (16.8 mm) in the horizontal direction, and the freeze-drying container 100 is provided with five storage units 50 that are not in communication with each other. Furthermore, the container body 10 was integrated with the first heat transfer body 30 . In addition, the entire freeze-drying container 100 was subjected to hydrophobic plating (eutectoid film of fluororesin and nickel, thickness: 10 ⁇ m) using “Nif Grip” (registered trademark) manufactured by ULVAC TECHNO.
  • Example 3 [Freeze-drying container] An aluminum alloy (A5052) with an outer dimension of 115 mm (length) x 115 mm (width) x 31 mm (height) was used, and an inner dimension of 105 mm (length) x 105 mm (width) x 28 mm (depth) was used. ) of the container body (second heat conductor, wall thickness: 5 mm, bottom thickness: 3 mm) and five plate-shaped first heat conductors (105 mm (vertical) x 3 mm (thickness) perpendicular to the bottom of the container body) (height) x 25 mm (height)).
  • the wall of the container body and the first heat conductor are arranged at equal intervals (15 mm) in the horizontal direction, and the freeze-drying container is provided with six storage units that are not in communication with each other, and further, The container body was integral with the first heat transfer body.
  • hydrophobic plating eutectoid film of fluororesin and nickel, thickness: 10 ⁇ m
  • Nif Grip registered trademark
  • Example 4 [Freeze-drying container] Using an aluminum alloy (A5052) with an outer dimension of 106 mm (length) x 106 mm (width) x 31 mm (height), a container body with an inner dimension of 96 mm (length) x 96 mm (width) x 28 mm (depth) second heat conductor, wall thickness: 5 mm, bottom thickness: 3 mm) and six plate-shaped first heat conductors perpendicular to the bottom of the container body (96 mm (length) ⁇ 3 mm (thickness) ⁇ 25 mm (height) A freeze-drying container was prepared, including The wall of the container body and the first heat transfer element are arranged at equal intervals (11.1 mm) in the horizontal direction, and the freeze-drying container is provided with seven storage units that are not in communication with each other, Furthermore, the container body was integral with the first heat transfer body.
  • a freeze-drying container was prepared, including The wall of the container body and the first heat transfer element are arranged at
  • a hydrophobic coating (eutectoid film of fluororesin and nickel, thickness: 10 ⁇ m) was applied to the entire freeze-drying container using “Nif Grip” (registered trademark) manufactured by ULVAC TECHNO.
  • Example 5 [Freeze-drying container] Using an aluminum alloy (A5052) with an outer dimension of 106 mm (length) x 106 mm (width) x 31 mm (height), a container body with an inner dimension of 96 mm (length) x 96 mm (width) x 28 mm (depth) Second heat conductor, wall thickness: 5 mm, bottom thickness: 3 mm) and seven plate-shaped first heat conductors perpendicular to the bottom of the container body (96 mm (length) ⁇ 3 mm (thickness) ⁇ 25 mm (height) A freeze-drying container was prepared, including The wall of the container body and the first heat transfer element are arranged at equal intervals (9.4 mm) in the horizontal direction, and the freeze-drying container is provided with eight storage units that are not in communication with each other, Furthermore, the container body was integral with the first heat transfer body.
  • a freeze-drying container was prepared, including The wall of the container body and the first heat transfer element are arranged
  • hydrophobic plating eutectoid film of fluororesin and nickel, thickness: 10 ⁇ m
  • Nif Grip registered trademark
  • Example 2 [Liquid to be freeze-dried, freeze-drying process] Using the same aqueous gelatin solution as in Example 1, 6.4 g of the aqueous gelatin solution was poured into each of the eight storage portions of the freeze-drying container. The volume of 6.4 g gelatin aqueous solution at 23° C. was 6.3 mL, measured in the same manner as in Example 1. Place the freeze-drying container in a freeze dryer (manufactured by ULVAC, DFR-5N-B), allow to stand at 7°C for 2 hours, cool to -10°C, then -30°C for 14 hours, then -10. C. for 44 hours and then vacuum drying at 20.degree.
  • a freeze dryer manufactured by ULVAC, DFR-5N-B
  • Example 6 [Freeze-drying container] A freeze-drying container was prepared in the same manner as in Example 5, except that the hydrophobic coating with Nif Grip (registered trademark) was not applied. body) was produced.
  • Example 7 [Freeze-drying container] Using an aluminum alloy (A5052) with an outer dimension of 93 mm (length) x 93 mm (width) x 31 mm (height), a container body (with an inner dimension of 83 mm (length) x 83 mm (width) x 28 mm (depth)) second heat conductor, wall thickness: 5 mm, bottom thickness: 3 mm) and one plate-shaped first heat conductor perpendicular to the bottom of the container body (83 mm (length) ⁇ 3 mm (thickness) ⁇ 25 mm (height) A freeze-drying container was prepared, including The wall of the container body and the first heat conductor are arranged at equal intervals (40.0 mm) in the horizontal direction, and the freeze-drying container is provided with two storage units that are not in communication with each other, Furthermore, the container body was integral with the first heat transfer body.
  • a freeze-drying container was prepared, including The wall of the container body and the first heat conductor are arranged
  • hydrophobic plating eutectoid film of fluororesin and nickel, thickness: 10 ⁇ m
  • Nif Grip registered trademark
  • Pore uniformity was evaluated in the following manner. Specimens were prepared for pore observation of the freeze-dried product. Specifically, first, the freeze-dried product was cut into a size of 1 cm ⁇ 1 cm, and thermally crosslinked by treating at 135° C. for 5 hours in a nitrogen atmosphere. Next, the resulting crosslinked product was immersed in physiological saline for 12 hours, and then embedded in an embedding agent for preparing frozen tissue sections (“OCT Compound” manufactured by Sakura Fine Tech Japan). Frozen at -20°C. Sections were obtained by slicing the resulting frozen block into slices with a thickness of about 5 ⁇ m using a cryostat.
  • OCT Compound manufactured by Sakura Fine Tech Japan
  • the section was attached to a slide glass, air-dried with cold air from a dryer for 5 minutes, and then immersed in an eosin staining solution for 1 minute to stain the section of the section.
  • the section was washed with water, dehydrated by immersion in ethanol, cleared by immersion in xylene, sealed with marinol and a cover glass, and dried at room temperature for 2 hours to obtain a specimen.
  • a 2.5 mm ⁇ 2.5 mm area of the sample was observed with an optical microscope to obtain the optical microscope images shown in FIGS. 9 to 15, the direction of the arrow indicates the in-plane direction of the liquid surface of the freeze-dried liquid, which is the precursor of the freeze-dried product.
  • Evaluation criteria are as follows. Practical levels are S and P. -Evaluation criteria- S: The pores are uniform and there are no protuberances in the liquid surface direction. P: The pores are almost uniform, but small bumps are observed in the direction of the liquid surface.
  • F Pores are uneven.
  • Table 1 shows the evaluation results of dryness, pore uniformity, and releasability.
  • freeze-drying containers of Examples 1 to 7 As shown in Table 1, freeze-dried products with uniform pores could be obtained.
  • Example 1 to 4 the first shortest distance and the second shortest distance were 5 mm or more. Moreover, in Examples 1 to 4, the ratio of the wetted area to the liquid volume was 200 mm 2 /mL or more. Therefore, Examples 1 to 4 were superior in pore uniformity. Examples 1 to 4 in which the first shortest distance and the second shortest distance are 5 mm or more are compared to Examples 5 to 6 in which the first shortest distance and the second shortest distance are less than 5 mm. , it is found that the pore uniformity is better.
  • Examples 1 to 4 in which the ratio of the wetted area to the liquid volume is 200 mm 2 /mL or more, are compared with Example 7, in which the ratio of the wetted area to the liquid volume is less than 200 mm 2 /mL. As a result, it can be seen that the pore uniformity is better.
  • Example 1 the ratio of the wetted area to the liquid volume was 500 mm 2 /mL or less, and thus the peelability was superior to that of Example 3. .
  • Example 5 which includes a hydrophobic coating, is superior in releasability.
  • Comparative Example 1 had non-uniform pores because it did not have a heat transfer body other than the container body.
  • Comparative Example 2 did not have a heat conductor other than the main body of the container, and because the amount of aqueous gelatin solution was large, it could not be completely dried, leaving an undried portion.

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