WO2001047544A1 - Protein-prefilled syringe preparation and method of stabilizing the same - Google Patents

Protein-prefilled syringe preparation and method of stabilizing the same Download PDF

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Publication number
WO2001047544A1
WO2001047544A1 PCT/JP2000/009309 JP0009309W WO0147544A1 WO 2001047544 A1 WO2001047544 A1 WO 2001047544A1 JP 0009309 W JP0009309 W JP 0009309W WO 0147544 A1 WO0147544 A1 WO 0147544A1
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WO
WIPO (PCT)
Prior art keywords
protein
prefilled syringe
resin
csf
preparation
Prior art date
Application number
PCT/JP2000/009309
Other languages
French (fr)
Japanese (ja)
Inventor
Yoshimi Imaeda
Daisuke Kameoka
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
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.)
Filing date
Publication date
Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to AU22275/01A priority Critical patent/AU2227501A/en
Publication of WO2001047544A1 publication Critical patent/WO2001047544A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/31—Details
    • A61M5/315—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms
    • A61M5/31511—Piston or piston-rod constructions, e.g. connection of piston with piston-rod
    • A61M5/31513—Piston constructions to improve sealing or sliding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/31—Details
    • A61M5/315—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms
    • A61M5/31511—Piston or piston-rod constructions, e.g. connection of piston with piston-rod
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/02—General characteristics of the apparatus characterised by a particular materials
    • A61M2205/0222—Materials for reducing friction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/02—General characteristics of the apparatus characterised by a particular materials
    • A61M2205/0238—General characteristics of the apparatus characterised by a particular materials the material being a coating or protective layer

Definitions

  • the present invention relates to a protein prefilled syringe preparation and a method for stabilizing the same.
  • the present invention relates to a protein prefilled syringe preparation which is easy to handle and stable for a long period of time. More specifically, the present invention relates to a stable protein prefilled syringe preparation comprising an elastic body coated with a resin on at least a protein solution contact surface. The invention further relates to a method for stabilizing a protein prefilled syringe formulation. Viewing technology
  • protein prefilled syringe preparations contain diluents, solubilizers, tonicity agents, excipients, pH adjusters, buffers, sulfur-containing reducing agents, antioxidants, etc., in addition to the active ingredient protein. And provided in the market in a syringe. Satisfactory conditions for protein prefilled syringe preparations include (1) that protein products are stable under long-term storage conditions, (2) that they have heat resistance and pressure resistance that can withstand the conditions used for sterilization, 3) It has chemical resistance, (4) it has excellent sealing properties, and (5) it has good plunger slidability.
  • a syringe has been required to have a hermetic seal, and in order to satisfy this requirement, a rubber sealing stopper made of a single rubber material having the highest hermeticity has been used.
  • the solution formulation and the rubber stopper are always in contact with the drug in the vial formulation, and the chemical effect of the drug from the rubber stopper is large, resulting in deterioration of the drug product.
  • maintaining long-term stability is difficult. Therefore, there is a demand for the development of a protein prefilled syringe formulation that can be stored for a long period of time, but a product that satisfies all the above requirements has not been developed. Disclosure of the invention
  • the present inventors have studied the above problems from various factors, and have found that a rubber stopper of a syringe used for a prefilled syringe formulation has an adverse effect on protein stabilization.
  • a rubber stopper of a syringe used for a prefilled syringe formulation has an adverse effect on protein stabilization.
  • there are variations in the surface treatment of rubber stoppers which greatly affects the residual rate of protein solution preparations depending on the production lot of rubber stoppers, and the variation in residual rates among individuals within the same lot is large. I found that.
  • the present inventors have found that laminating a rubber stopper with Teflon can greatly contribute to the improvement of protein stability, particularly when the protein is granulocyte colony stimulating factor (G-CSF). That is, the present inventors have completed the present invention by finding that when a plastic film is laminated on the surface of a stopper made of a rubber material alone, a high content of protein can be retained for an extremely long time.
  • G-CSF granulocyte colony stimulating factor
  • the present invention provides a stable protein prefilled syringe preparation comprising an elastic body coated with a resin on at least the protein solution contact surface.
  • the present invention provides the above-mentioned protein prefilled syringe preparation, comprising a stopper having a resin film laminated on at least a protein solution contact surface.
  • the present invention provides the protein prefilled syringe preparation, wherein the resin is selected from polyethylene, polypropylene, polycarbonate, polyester, and fluororesin. I will provide a.
  • the present invention provides the protein prefilled syringe preparation, wherein the resin is a fluororesin.
  • the present invention provides the above protein prefilled syringe formulation, wherein the resin is polytetrafluoroethylene.
  • the present invention provides the above-mentioned protein pre-filled syringe preparation, wherein the protein is a recombinant protein.
  • the present invention provides the above protein prefilled syringe formulation, wherein the protein is erythropoietin.
  • the present invention provides the above-mentioned protein pre-filled syringe formulation, wherein the protein is a granulocyte colony stimulating factor.
  • the present invention provides the above protein pre-filled syringe preparation, wherein the protein is a protein having a sugar chain.
  • the present invention provides a method for stabilizing a protein prefilled syringe formulation, which comprises at least filling and storing a protein solution formulation in a syringe provided with an elastic body coated with a resin on the protein solution contact surface.
  • the material of the syringe used in the present invention may be a material known in the art, and may be glass or resin.
  • Suitable resins include polymethylpentene resin, polypropylene resin, propylene-ethylene copolymer resin, polyester resin, polyethersulfone resin, polysulfone resin, or any of the above resins and polyamide resin, polyvinyl alcohol resin, ethylene vinyl acetate. It is preferable to use a resin obtained by laminating two or more layers of a resin selected from a resin and a resin.
  • Particularly preferred resin materials for protein solution preparations include those described in Japanese Patent Application No. 11-254896.
  • PE polyethylene
  • PP polypropylene
  • PET polyethylene terephthalate
  • PCT polycarbonate
  • polyethylene methacrylate is preferably made of, for example, silicone such as lacyclododecene or a derivative thereof.
  • Cyclopentyl residue or substituted cyclopentyl residue was inserted into the molecular chain by polymerization of ethylene- or propylene with ring-opening polymer and its hydrogenated product, cycloolefin such as norbornene or tetracyclododecene or its derivative, and ethylene or propylene. It is a resin that is a copolymer.
  • the cycloolefin includes monocyclic and polycyclic ones.
  • Preferred is a thermoplastic norportene resin or a thermoplastic tetracyclododecene resin.
  • examples of the thermoplastic norbornene-based resin include a ring-opened polymer of a norbornene-based monomer, a hydrogenated product thereof, an addition-type polymer of a norbornene-based monomer, and an addition-type polymer of a norbornene-based monomer and an olefin. I can do it.
  • thermoplastic tetracyclododecene resin examples include a ring-opened polymer of a tetracyclododecene monomer, a hydrogenated product thereof, an addition polymer of a tetracyclododecene monomer, and a tetracyclododecene monomer.
  • An addition type polymer of a decene monomer and an olefin is exemplified.
  • the elastic body used in the present invention is used as a stopper (slider) of a syringe and a rubber stopper for sealing, and is preferably made of a rubber body.
  • the elastic body can also be manufactured using any known rubber elastic body used for pharmaceutical containers and medical equipment, and further copolymerized with natural rubber, isoprene rubber, butadiene rubber, butyl rubber, and divinyl benzene.
  • thermoplastic elastomer As a thermoplastic elastomer, there is a technique of using a styrene-butadiene copolymer and a hydrogenated product of the copolymer as a plug for a pharmaceutical container (Japanese Patent Application Laid-Open No. 59-289695, Publication No. 57-266782, Japanese Patent Publication No. Hei 2-424296). Also, a technology in which a mixture of a thermoplastic elastomer and an isoprene-isobutylene copolymer is used as a rubber stopper (Japanese Patent Application Laid-Open No.
  • a protein solution contact surface of the elastic body is coated with a resin.
  • a coating method various methods known to those skilled in the art, such as lamination (lamination) with a resin film or spraying (coating) of a resin, can be used.
  • the resin used in the present invention any resin which is inert to the protein solution preparation filled in the prefilled syringe can be used.
  • polyethylene, polypropylene, polycarbonate, polyester, fluororesin and the like can be mentioned.
  • fluororesins such as polytetrafluoroethylene (PTF E: Teflon (registered trademark)), tetrafluoroethylene-ethylene copolymer (E TFE), and tetrafluoroethylene-hexane.
  • PPF E Teflon (registered trademark)
  • E TFE tetrafluoroethylene-ethylene copolymer
  • tetrafluoroethylene-hexane tetrafluoroethylene-hexane.
  • PCTFE polychloro trifluoroethylene
  • PTFE polytetrafluoroethylene
  • These resins are preferably used as a sheet or a film, and the thickness is not particularly limited, but
  • the elastic body used for the protein prefilled syringe preparation of the present invention is usually produced by compression molding using the above rubber composition and a resin film.
  • a stopper is disclosed in, for example, JP-A-10-248929 and JP-A-10-314305.
  • Proteins used as an active ingredient in the present invention include, for example, hematopoietic factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), erythropoietin (EP ⁇ ), and tropopoetin; Cytochromes such as IL-1 and IL-6, monoclonal antibodies, tissue plasminogen-activating factor (TPA), perokinase, serum albumin, blood coagulation factor VIII, lebutin, insulin , Including but not limited to, stem cell growth factor (SCF).
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • EP ⁇ erythropoietin
  • TPA tissue plasminogen-activating factor
  • SCF stem cell growth factor
  • hematopoietic factors such as EP ⁇ , G-CSF, and thrombop
  • the protein used as an active ingredient in the present invention is a protein having substantially the same biological activity as that of a biologically active protein of a mammal, particularly a human, and is obtained from a naturally-occurring protein or a recombinant gene. Preferred are those obtained by the gene recombination method.
  • the protein obtained by the genetic recombination method is unstable compared to the natural protein, and it is preferable to use the preparation of the present invention.
  • Proteins obtained by the genetic recombination method include those having the same amino acid sequence as the natural protein, or those having one or more of the amino acid sequences deleted, substituted, or added and having the biological activity. Including.
  • proteins include those chemically modified with PEG and the like.
  • Examples of the protein used as an active ingredient in the present invention include a protein having a sugar chain.
  • the origin of the sugar chain is not particularly limited, but a sugar chain added to mammalian cells is preferable.
  • Mammalian cells include, for example, Chinese hamster ovary cells (CHO cells), BHK cells, COS cells, human-derived cells and the like, among which CHO cells are most preferred.
  • EP ⁇ may be produced by any method, extracted from human urine by various methods, separated and purified, using a genetic engineering technique (for example, according to Japanese Unexamined Patent Publication No. Sho 61-12288), hamster ovary cells (CHO), BHK cells, COS cells, human-derived cells, and the like, which are extracted and separated and purified by various methods, are used. Furthermore, it includes EPO chemically modified by PEG or the like (see International Patent Application Publication No. W090 / 12874). Furthermore, EPO with no sugar chain is chemically modified with PEG or the like.
  • EPO analogs that have been modified to increase the number of one or more glycosylation sites at the N-linked carbohydrate chain binding site or the O-linked carbohydrate chain binding site in the amino acid sequence of EPO (eg, (See Kaihei 8—151398, Tokuhei 8-506023). Further, the amount of sugar chains may be increased by increasing the content of sialic acid or the like without changing the number of sugar chain binding sites.
  • any G-CSF can be used as long as it is highly purified G-CSF.
  • the G-CSF in the present invention may be produced by any method, such as those obtained by culturing a cell line of human tumor cells and extracting and separating and purifying it from various cells, or E. coli by genetic engineering techniques. Bacteria; Listeria monocytogenes; Chinese hamster ovary (CH ⁇ ) cells, C127 cells, COS cells, and other animal-derived culture cells that are extracted and purified by various methods. Can be Like Alternatively, it is produced by E. coli, yeast, or CHO cells using a genetic recombination method. Most preferably, it is produced by a genetic modification method using CH ⁇ ⁇ ⁇ cells. Furthermore, G-CSF chemically modified with PEG or the like is also included (see International Patent Application Publication No. WO 90/12874).
  • the monoclonal antibody may be produced by any method.
  • Monoclonal antibodies are basically immunized with a sensitizing antigen according to a usual immunization method using a known technique, and the obtained immune cells are fused with a known parent cell by a normal cell fusion method.
  • the monoclonal antibody is not limited to the monoclonal antibody produced by the hybridoma, and includes a chimeric antibody artificially modified for the purpose of, for example, reducing the xenoantigenicity to humans.
  • a reshaped (reshaped) humanized antibody can also be used in the present invention. This is obtained by replacing the complementarity-determining region of a human antibody with the complementarity-determining region of a non-human mammal, for example, a mouse antibody, and a general gene recombination technique is also known. Using the known method, a reshaped humanized antibody useful in the present invention can be obtained.
  • a reshaped humanized antibody is a humanized PM-1 (hPM-1) antibody which is an anti-IL-16 receptor humanized antibody (International Patent Application Publication No. WO 92-19759).
  • the antibodies of the present invention also include human antibodies produced by known techniques using transgenic animals and the like.
  • the present invention can be applied to antibody fragments (for example, Fab, F (ab) 2, etc.) and modified antibodies (for example, single-chain antibodies, etc.).
  • antibody fragments for example, Fab, F (ab) 2, etc.
  • modified antibodies for example, single-chain antibodies, etc.
  • chimeric antibodies, humanized antibodies, antibody fragments, and modified antibodies are more unstable than natural antibodies, and therefore it is preferable to use the formulations of the present invention.
  • the protein preparation of the present invention may contain a diluent, a solubilizing agent, a tonicity agent, an excipient, a pH adjuster, a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, etc. Good.
  • a solubilizing agent polyethylene glycol; dextran, mannitol, sorbitol, inositol, glucose, flak, and lac!
  • You can use sugars such as sucrose, xylose, mannose, maltose, sucrose, and raffinose.
  • sulfur-containing reducing agent examples include N-acetyl cysteine, N-acetyl homocystine, thioctic acid, thiodiglycol, thioethanolamine, thiodaricerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, and dal.
  • sulfur-containing reducing agent examples include N-acetyl cysteine, N-acetyl homocystine, thioctic acid, thiodiglycol, thioethanolamine, thiodaricerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, and dal.
  • sulfur-containing reducing agent examples include N-acetyl cysteine, N-acetyl homocystine, thioctic acid, thiodiglycol, thioethanolamine, thiodaricerol, thiosorbitol,
  • antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, and L-ascorbic acid Chelating agents such as stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or disodium ethyl diamine tetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate and the like.
  • EDTA disodium ethyl diamine tetraacetate
  • inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium hydrogen carbonate
  • solution preparations such as organic salts such as sodium citrate, potassium citrate, and sodium acetate. It may contain components that are usually added.
  • the protein solution preparation of the present invention may further contain an appropriate stabilizer for each protein, and the stabilizer includes a surfactant (for example, sorbitan fatty acid ester or glycerin fatty acid ester which is a nonionic surfactant).
  • a surfactant for example, sorbitan fatty acid ester or glycerin fatty acid ester which is a nonionic surfactant.
  • the stable protein prefilled syringe preparation of the present invention is usually administered by parenteral administration route, for example, injection (subcutaneous or intravenous injection), transdermal, transmucosal, nasal, etc., but oral administration is also possible.
  • parenteral administration route for example, injection (subcutaneous or intravenous injection), transdermal, transmucosal, nasal, etc., but oral administration is also possible.
  • the amount of protein contained in the stable protein prefilled syringe formulation of the present invention can be determined according to the protein used, the type of disease to be treated, the severity of the disease, the age of the patient, and the like.
  • 0.01 g / m1 or more, preferably 0.1 gZm1 or more, more preferably 1 gZm1 or more, more preferably 10 / ig Zm1 or more, further preferably 50 g / m Contains 1 or more proteins.
  • a prefilled syringe solution preparation it contains 0.1 gZml or more, preferably 1 / xg noml or more, more preferably 10 g / m1 or more, and still more preferably 50 gZm1 or more.
  • the amount of EP ⁇ in the solution formulation is generally between 100 and 500 000 I UZm, preferably between 200 and 100000 I UZm and more preferably between 750 and 72000 1 U / m 1.
  • G-CSF it is generally l to 1000 g / m 1, preferably 10 to 800 // g Zm, and more preferably 50 to 500 / ig / m 1.
  • the final administration concentration is generally 0.1 to 20 OmgZm, preferably 1 to 20 OmgZm. ⁇ 12 Omg / m1.
  • the protein prefilled syringe formulation of the present invention can be prepared by dissolving these components in an aqueous buffer known in the field of solution formulation, such as phosphate and Z or citrate buffers.
  • the phosphate buffer is preferably a sodium monohydrogen phosphate sodium dihydrogen phosphate system
  • the citrate buffer is preferably a sodium citrate buffer solution.
  • a buffer such as a glycine buffer or a histidine buffer may be used.
  • the evening protein prefilled syringe preparation of the present invention is a erythrocyte poetin solution preparation
  • a nonionic surfactant for example, polysorbate 80, polysorbate 20
  • an isotonic agent It preferably contains, for example, sodium chloride
  • a stabilizer eg, an amino acid, preferably L-histidine
  • the preparation contains G-CSF, a nonionic surfactant (for example, polysorbate 80, polysorbate 20), and if necessary, a diluent.
  • G-CSF a nonionic surfactant
  • the anti-IL16 receptor humanized antibody is preferably used in a glycine buffer and a Z or histidine buffer. Dissolved in a buffer solution such as a liquid, and other nonionic surfactants (for example, polysorbate 80, polysorbate 20), and if necessary, diluents, dissolution aids, isotonic agents, excipients, and pH adjusters. It preferably contains a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, and the like, and has a pH of 5 to 8.
  • a buffer solution such as a liquid, and other nonionic surfactants (for example, polysorbate 80, polysorbate 20), and if necessary, diluents, dissolution aids, isotonic agents, excipients, and pH adjusters. It preferably contains a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, and the like, and has a pH of 5 to 8.
  • a stable protein pre-filled syringe formulation pre-filled in a syringe equipped with a stopper in which the plastic film of the present invention was laminated at least on the protein solution contact surface was tested using G-CSF, as shown in Examples described later. Even after a 2-week accelerated test at 40 ° C, the G-CSF retention rate is much better than that of a non-laminated stopper. Or 6 months at 25 ° C, 1 Even after long-term storage at 0 ° C for 1 year, it shows good G-CSF retention. Also, if the protein is EPO, even after accelerated tests at 50 ° C-2 weeks or 40 ° C-1 month, or at 6 months at 25 ° C and 1 year at 10 ° C Very good EPO persistence after storage.
  • the present invention also provides a method for stabilizing a protein prefilled syringe formulation, which comprises filling and storing a protein solution formulation in a syringe provided with a stopper having a plastic film laminated on at least the protein solution contact surface.
  • the stabilization in the present invention depends on the type of protein to be filled. For example, in the case of a G-CSF solution preparation (containing 12 G-CSF), this is stored at, for example, 40 ° C for 2 weeks or more. At that time, it means that the residual ratio of G-CSF is maintained at 75% or more, preferably 80% or more, and more preferably 85% or more.
  • the residual ratio of G-CSF is 80% or more, preferably 85% or more, more preferably Means to keep it above 90%.
  • a protein prefilled syringe preparation can be stably stored at room temperature for a long period of time.
  • a stable protein prefilled syringe formulation comprising a stopper in which the plastic film of the present invention is laminated at least on the protein solution contacting surface is a stable protein prefilled syringe formulation. More stable than syringe formulations.
  • a protein prefilled syringe preparation can be stably stored at room temperature for a long period of time.
  • the protein is obtained by a genetic recombination method, it is unstable compared to natural proteins, and should be stably stored at room temperature for a long period of time by using the prefilled syringe preparation of the present invention.
  • G-CSF granulocyte colony stimulating factor
  • the evaluation of the preparation was performed by determining the content of G-CSF using RP-HPLC analysis.
  • Example 1 G-CSF solution at 40 ° C for 2 weeks accelerated test
  • the G-CSF used in this example is a recombinant protein having a sugar chain (rG-CSF) produced in CHO cells.
  • Container form a recombinant protein having a sugar chain (rG-CSF) produced in CHO cells.
  • a PTFE-laminated glass syringe is filled with the above dispensing solution, and the stopper is sealed and stoppered by attaching a PTFE sheet to the liquid contacting surface of a WEST stop stopper (Material: bromobutyl rubber PH4023 / 50). .
  • Untreated stopper A syringe filled with the above preparation liquid and sealed with a stopper (material: bromobutyl rubber PH4023 / 50) manufactured by WEST.
  • PTFE laminating stock A PTFE sheet with a PTFE sheet attached to the liquid contacting surface of a WEST stopper (material: PTFE is peeled from bromobutyper), and the liquid contacting surface is used. Was peeled off. A glass syringe filled with the above dispensing solution and sealed and stoppered using this stopper.
  • Example 2 Storage stability test of G-CSF solution formulation at 25 ° C and 10 ° C
  • each container form containing the solution preparation prepared by aseptic preparation and filtration was stored at 25 ° C and 10 ° C for a long time. Thereafter, the residual ratio was calculated in the same manner as in Example 1. Tables 3 and 4 show the results.
  • EPO solution preparations of the following two concentrations (1500 IUZmL and 48000 IU / mL) were prepared, filled aseptically into containers equipped with PTFE laminate stoppers (BD), and stoppered. Accelerated tests were performed at 50 ° C and 40 ° C, and long-term storage tests were performed at 25 ° C and 10 ° C, and the residual rates were calculated respectively. The results are shown in Table 5-8. Formulation: 1500IU L 48000IU / mL

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  • Engineering & Computer Science (AREA)
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Abstract

A stabilized protein-prefilled syringe preparation equipped with a stopper which has a plastic film laminated at least to the surface in contact with the protein solution.

Description

明細書  Specification
タンパク質プレフィルドシリンジ製剤およびその安定化方法 技術分野  TECHNICAL FIELD The present invention relates to a protein prefilled syringe preparation and a method for stabilizing the same.
本発明は取り扱いが簡単で長期安定なタンパク質プレフィルドシリンジ製剤 に関する。 さらに詳しくは、本発明は少なくともタンパク質溶液接触面に樹脂を 被覆した弾性体を具備した、安定したタンパク質プレフィルドシリンジ製剤に関 する。本発明はさらにタンパク質プレフィルドシリンジ製剤の安定化方法に関す る。 向景技術  The present invention relates to a protein prefilled syringe preparation which is easy to handle and stable for a long period of time. More specifically, the present invention relates to a stable protein prefilled syringe preparation comprising an elastic body coated with a resin on at least a protein solution contact surface. The invention further relates to a method for stabilizing a protein prefilled syringe formulation. Viewing technology
遺伝子組換え技術の発達によって、種々のタンパク質製剤が安定した供給量で 提供されるようになった。 これらの製剤は安定性を確保するため、凍結乾燥した タンパク質成分粉末とこれを溶解するための水溶性希釈液とを別途包装し、使用 時に溶解する形態で提供されるか、あるいは安定性を向上させるための添加剤を 加えたタンパク質溶液製剤の形で提供されている。両製剤を比較するとき、使用 時の便宜性を考えると、 溶液製剤が有利である。 特に、 デイスポーザブル注射器 に予め溶液を充填して、プレフィルドシリンジ溶液製剤として供給することによ り、 溶解操作や薬液吸引の手間が省け、 迅速な対応が可能となる。 しかしながら、 溶液製剤の場合には、 安定性を確保することが困難である。  With the development of genetic recombination technology, various protein preparations have been provided in a stable supply amount. To ensure stability, these preparations are packaged separately with lyophilized protein component powder and a water-soluble diluent to dissolve it, and are provided in a form that can be dissolved at the time of use, or have improved stability It is provided in the form of a protein solution formulation to which additives are added. When comparing the two formulations, the solution formulation is advantageous because of the convenience in use. In particular, by dispensing a disposable syringe in advance and supplying it as a prefilled syringe solution preparation, the time required for dissolution operation and aspiration of the drug solution can be reduced, and quick response can be achieved. However, in the case of solution preparations, it is difficult to ensure stability.
通常、 タンパク質プレフィルドシリンジ製剤は、有効成分であるタンパク質に 加えて、 希釈剤、 溶解補助剤、 等張化剤、 賦形剤、 P H調整剤、 緩衝剤、 含硫還 元剤、 酸化防止剤などを含み、 これを注射器中に入れて市場に提供される。 タン パク質プレフィルドシリンジ製剤が満足すべき条件として、 （1 ) タンパク質製 剤が長期保存状態で安定であること、 （2 ) 滅菌に使用する条件に耐えうる耐熱 性、 耐圧性を有すること、 （3 ) 耐薬品性を有すること、 （4 ) 密封性に優れて いること、 （5 ) プランジャーの摺動性がよいこと、 などが挙げられる。  Normally, protein prefilled syringe preparations contain diluents, solubilizers, tonicity agents, excipients, pH adjusters, buffers, sulfur-containing reducing agents, antioxidants, etc., in addition to the active ingredient protein. And provided in the market in a syringe. Satisfactory conditions for protein prefilled syringe preparations include (1) that protein products are stable under long-term storage conditions, (2) that they have heat resistance and pressure resistance that can withstand the conditions used for sterilization, 3) It has chemical resistance, (4) it has excellent sealing properties, and (5) it has good plunger slidability.
タンパク質をプレフィルドシリンジ製剤とするときに、長期保存しょうとする 場合にはタンパク質の残存率の低下が生じ、安定性が必ずしも十分ではなかった このような安定性欠如の要因の一つとして、プレフィルドシリンジ製剤に使用す るディスポ一ザブル注射器のストッパーの材質が挙げられる。 When preserving protein for a long period of time when preparing a prefilled syringe, the residual ratio of the protein decreased, and the stability was not always sufficient One of the factors of such a lack of stability is the material of the stopper of the disposable syringe used for the prefilled syringe preparation.
従来から、注射器では密封性が要求され、 この要求を満たすためには最も密封 性に優れたゴム素材単体のゴム製密封栓が使用されてきた。 しかし、 プレフィル ドシリンジ注射器では、バイアル製剤中の薬剤と比べて、溶液製剤とゴム栓とが 常に接触しており、 このため薬剤のゴム栓から受ける化学的影響が大きく、 医薬 品の変質が起こり、 長期的な安定性の維持が困難なことが懸念されている。 従って、長期保存できるタンパク質プレフィルドシリンジ製剤の開発が望まれ ているが、 上記の要件を全て満足するものは開発されていない。 発明の開示  Conventionally, a syringe has been required to have a hermetic seal, and in order to satisfy this requirement, a rubber sealing stopper made of a single rubber material having the highest hermeticity has been used. However, in a prefilled syringe, the solution formulation and the rubber stopper are always in contact with the drug in the vial formulation, and the chemical effect of the drug from the rubber stopper is large, resulting in deterioration of the drug product. There is concern that maintaining long-term stability is difficult. Therefore, there is a demand for the development of a protein prefilled syringe formulation that can be stored for a long period of time, but a product that satisfies all the above requirements has not been developed. Disclosure of the invention
本発明者らは上記課題を種々の要因から検討した結果、プレフィルドシリンジ 製剤に使用する注射器のゴムストッパーがタンパク質安定化に悪影響を及ぼす ことを発見した。 特に、 ゴムストッパーの表面処理にバラツキがあり、 このため ゴムストッパーの製造ロットによってタンパク質溶液製剤の残存率が大きく影 響されること、さらに同一ロット内においても個体間で残存率のバラツキが大き いことを見出した。  The present inventors have studied the above problems from various factors, and have found that a rubber stopper of a syringe used for a prefilled syringe formulation has an adverse effect on protein stabilization. In particular, there are variations in the surface treatment of rubber stoppers, which greatly affects the residual rate of protein solution preparations depending on the production lot of rubber stoppers, and the variation in residual rates among individuals within the same lot is large. I found that.
本発明者らは、 特にタンパク質が顆粒球コロニー刺激因子 （G - CSF) である場 合に、 ゴムストッパーをテフロンでラミネートすることにより、 タンパク質の安 定性向上に大きく寄与しうることを発見した。 すなわち、 本発明者らは、 ゴム素 材単独から製造されるストッパー表面にプラスチックフィルムをラミネ一トす ると極めて長期にわたって高含量のタンパク質を保持できることを見いだして 本発明を完成した。  The present inventors have found that laminating a rubber stopper with Teflon can greatly contribute to the improvement of protein stability, particularly when the protein is granulocyte colony stimulating factor (G-CSF). That is, the present inventors have completed the present invention by finding that when a plastic film is laminated on the surface of a stopper made of a rubber material alone, a high content of protein can be retained for an extremely long time.
すなわち、本発明は、少なくともタンパク質溶液接触面に樹脂を被覆した弾性 体を具備した、 安定したタンパク質プレフィルドシリンジ製剤を提供する。 本発明は、樹脂フィルムを少なくともタンパク質溶液接触面にラミネートした ストッパーを具備した、前記タンパク質プレフィルドシリンジ製剤を提供する。 本発明は、 樹脂がポリエチレン、 ポリプロピレン、 ポリカーボネート、 ポリエ ステル、フッ素系樹脂から選択される前記タンパク質プレフィルドシリンジ製剤 を提供する。 That is, the present invention provides a stable protein prefilled syringe preparation comprising an elastic body coated with a resin on at least the protein solution contact surface. The present invention provides the above-mentioned protein prefilled syringe preparation, comprising a stopper having a resin film laminated on at least a protein solution contact surface. The present invention provides the protein prefilled syringe preparation, wherein the resin is selected from polyethylene, polypropylene, polycarbonate, polyester, and fluororesin. I will provide a.
本発明は、樹脂がフッ素系樹脂である前記タンパク質プレフィルドシリンジ製 剤を提供する。  The present invention provides the protein prefilled syringe preparation, wherein the resin is a fluororesin.
本発明は、樹脂がポリテトラフルォロエチレンである前記タンパク質プレフィ ルドシリンジ製剤を提供する。  The present invention provides the above protein prefilled syringe formulation, wherein the resin is polytetrafluoroethylene.
本発明は、タンパク質が遺伝子組換え夕ンパク質である前記タンパク質プレフ ィルドシリンジ製剤を提供する。  The present invention provides the above-mentioned protein pre-filled syringe preparation, wherein the protein is a recombinant protein.
本発明は、タンパク質がエリスロポエチンである前記タンパク質プレフィルド シリンジ製剤を提供する。  The present invention provides the above protein prefilled syringe formulation, wherein the protein is erythropoietin.
本発明は、タンパク質が顆粒球コロニー刺激因子である前記タンパク質プレフ ィルドシリンジ製剤を提供する。  The present invention provides the above-mentioned protein pre-filled syringe formulation, wherein the protein is a granulocyte colony stimulating factor.
本発明は、タンパク質が糖鎖を有するタンパク質である前記タンパク質プレフ ィルドシリンジ製剤を提供する。  The present invention provides the above protein pre-filled syringe preparation, wherein the protein is a protein having a sugar chain.
本発明は、なくともタンパク質溶液接触面に樹脂を被覆した弾性体を具備した 注射器にタンパク質溶液製剤を充填して保存することからなるタンパク質プレ フィルドシリンジ製剤の安定化方法を提供する。 発明を実施するための最良の形態  The present invention provides a method for stabilizing a protein prefilled syringe formulation, which comprises at least filling and storing a protein solution formulation in a syringe provided with an elastic body coated with a resin on the protein solution contact surface. BEST MODE FOR CARRYING OUT THE INVENTION
本発明で使用する注射器の材料は、当該技術分野で公知のものを用いることが でき、 ガラスであっても樹脂であってもよい。適切な樹脂にはポリメチルペンテ ン樹脂、 ポリプロピレン樹脂、 プロピレン—エチレン共重合樹脂、 ポリエステル 樹脂、 ポリエーテルスルホン樹脂、 ポリスルホン樹脂、 あるいは上記樹脂とポリ アミド樹脂、ポリビニールアルコール樹脂、エチレン 酢酸ビニルの鹼化体から 選択される樹脂とを 2層以上に積層したものを用い、成形したものが好ましい。 タンパク質溶液製剤に特に好ましい樹脂材料としては、特願平 1 1— 2 5 4 8 9 6号に記載されているものを含む。 すなわち、 ポリエチレン （P E ) 、 ポリプロ ピレン （P P ) 、 ポリエチレンテレフ夕レート （P E T) 、 ポリカーボネート、 ポリェチルメタクリレートなどの公知の医用容器材料を含み、好ましいのは、例 ラシクロドデセンまたはその誘導体などのシク ン開環重合体およびその水素添加物、ノルボルネンもしくはテトラシ クロドデセンまたはその誘導体などのシクロォレフインと、エチレンまたはプロ ピレンとの重合により分子鎖にシクロべンチル残基や置換シクロべンチル残基 が挿入された共重合体である樹脂である。 ここで、 シクロォレフインは単環式お よび多環式のものを含む。好ましいのは、熱可塑性ノルポルネン系樹脂または熱 可塑性テトラシクロドデセン系樹脂である。熱可塑性ノルボルネン系樹脂として は、 ノルボルネン系単量体の開環重合体、 その水素添加物、 ノルボルネン系単量 体の付加型重合体、ノルボルネン系単量体とォレフィンの付加型重合体などが挙 げられる。熱可塑性テトラシクロドデセン系樹脂としては、 テトラシクロドデセ ン系単量体の開環重合体、その水素添加物、テトラシクロドデセン系単量体の付 加型重合体、テトラシクロドデセン系単量体とォレフィンの付加型重合体などが 挙げられる。 The material of the syringe used in the present invention may be a material known in the art, and may be glass or resin. Suitable resins include polymethylpentene resin, polypropylene resin, propylene-ethylene copolymer resin, polyester resin, polyethersulfone resin, polysulfone resin, or any of the above resins and polyamide resin, polyvinyl alcohol resin, ethylene vinyl acetate. It is preferable to use a resin obtained by laminating two or more layers of a resin selected from a resin and a resin. Particularly preferred resin materials for protein solution preparations include those described in Japanese Patent Application No. 11-254896. That is, it includes well-known medical container materials such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polycarbonate, and polyethylene methacrylate, and is preferably made of, for example, silicone such as lacyclododecene or a derivative thereof. Cyclopentyl residue or substituted cyclopentyl residue was inserted into the molecular chain by polymerization of ethylene- or propylene with ring-opening polymer and its hydrogenated product, cycloolefin such as norbornene or tetracyclododecene or its derivative, and ethylene or propylene. It is a resin that is a copolymer. Here, the cycloolefin includes monocyclic and polycyclic ones. Preferred is a thermoplastic norportene resin or a thermoplastic tetracyclododecene resin. Examples of the thermoplastic norbornene-based resin include a ring-opened polymer of a norbornene-based monomer, a hydrogenated product thereof, an addition-type polymer of a norbornene-based monomer, and an addition-type polymer of a norbornene-based monomer and an olefin. I can do it. Examples of the thermoplastic tetracyclododecene resin include a ring-opened polymer of a tetracyclododecene monomer, a hydrogenated product thereof, an addition polymer of a tetracyclododecene monomer, and a tetracyclododecene monomer. An addition type polymer of a decene monomer and an olefin is exemplified.
本発明で使用する弾性体は注射器のストッパー（滑栓）及び密封用ゴム栓とし て使用するものであり、好ましくはゴム体で製造される。弾性体は公知のいかな る医薬品容器、医療用具用器具類に使用するゴム弾性体を用いても製造すること ができ、 天然ゴム、 イソプレンゴム、 ブタジエンゴム、 ブチルゴム、 ジビニルべ ンゼンをさらに共重合したブチルゴム、塩素化又は臭素化ブチルゴム、エチレン 一プロピレン共重合ゴム、 イソプレン—イソブチレン共重合ゴム、 イソプレン— イソブチレン共重合体のハロゲン化ゴム、熱可塑性エラストマ一等を含む。熱可 塑性エラストマ一には、スチレン一ブタジエン共重合体並びに該共重合体の水素 添加物を医薬品容器の栓体とする技術がある（特開昭 5 9 - 2 8 9 6 5号公報、 特公昭 5 7— 2 6 7 8 2号公報、 特公平 2 - 4 2 9 6号公報） 。 また、 熱可塑性 エラストマ一とイソプレン—イソブチレン共重合体の混合物をゴム栓にした技 術 （特開昭 5 9 - 2 8 9 6 5号公報） 、 イソプレン一イソブチレン共重合体にフ ッ素ゴムを積層したゴム栓（特公昭 6 3 - 4 3 1 0 4号公報、実開昭 5 5— 4 7 8 5 0号公報） がある。  The elastic body used in the present invention is used as a stopper (slider) of a syringe and a rubber stopper for sealing, and is preferably made of a rubber body. The elastic body can also be manufactured using any known rubber elastic body used for pharmaceutical containers and medical equipment, and further copolymerized with natural rubber, isoprene rubber, butadiene rubber, butyl rubber, and divinyl benzene. Butyl rubber, chlorinated or brominated butyl rubber, ethylene-propylene copolymer rubber, isoprene-isobutylene copolymer rubber, isoprene-isobutylene copolymer halogenated rubber, thermoplastic elastomer and the like. As a thermoplastic elastomer, there is a technique of using a styrene-butadiene copolymer and a hydrogenated product of the copolymer as a plug for a pharmaceutical container (Japanese Patent Application Laid-Open No. 59-289695, Publication No. 57-266782, Japanese Patent Publication No. Hei 2-424296). Also, a technology in which a mixture of a thermoplastic elastomer and an isoprene-isobutylene copolymer is used as a rubber stopper (Japanese Patent Application Laid-Open No. 59-28965), and a fluoroelastomer is used for an isoprene-isobutylene copolymer. There is a laminated rubber stopper (Japanese Patent Publication No. 63-314104, Japanese Utility Model Publication No. 55-47950).
本発明では、上記弾性体の少なくともタンパク質溶液接触面に樹脂を被覆する。 被覆の方法は樹脂フィルムで積層 （ラミネート） する、 あるいは樹脂を噴霧 （コ 一ティング） するなど当業者に公知の種々の方法を用いることができる。 本発明で使用する樹脂は、プレフィルドシリンジに充填されるタンパク質溶液 製剤に対して不活性であるものは全て使用できる。 例えば、 ポリエチレン、 ポリ プロピレン、 ポリカーボネート、 ポリエステル、 フッ素系樹脂などが挙げられる。 特に好ましいのはフッ素系樹脂であり、 ポリテトラフルォロエチレン （PTF E :テフロン （登録商標） ） 、 テトラフルォロエチレン一エチレン共重合体 （E TFE) 、 テトラフルォロエチレン—へキサフルォロプロピレン共重合体 （FE P) 、 テトラフルォロエチレン一フルォロアルキルビニルエーテル共重合体（P FA) 、 ポリフッ化ビニリデン （PVDF) 、 ポリクロ口トリフルォロエチレン (PCTFE)などが挙げられる。最も好ましいのはポリテトラフルォロェチレ ン （PTFE) である。 これらの樹脂は、 好ましくはシート又はフィルムとして 用いられ、 厚さは特に限定されないが、 通常 1〜500 である。 In the present invention, at least a protein solution contact surface of the elastic body is coated with a resin. As a coating method, various methods known to those skilled in the art, such as lamination (lamination) with a resin film or spraying (coating) of a resin, can be used. As the resin used in the present invention, any resin which is inert to the protein solution preparation filled in the prefilled syringe can be used. For example, polyethylene, polypropylene, polycarbonate, polyester, fluororesin and the like can be mentioned. Particularly preferred are fluororesins, such as polytetrafluoroethylene (PTF E: Teflon (registered trademark)), tetrafluoroethylene-ethylene copolymer (E TFE), and tetrafluoroethylene-hexane. Fluoropropylene copolymer (FEP), tetrafluoroethylene-fluoroalkylvinylether copolymer (PFA), polyvinylidene fluoride (PVDF), polychloro trifluoroethylene (PCTFE), etc. Can be Most preferred is polytetrafluoroethylene (PTFE). These resins are preferably used as a sheet or a film, and the thickness is not particularly limited, but is usually 1 to 500.
本発明のタンパク質プレフィルドシリンジ製剤に使用する弾性体は、上記のゴ ム組成物と樹脂フィルムを用いて、通常圧縮成形によって製造される。 このよう なストツバ一は例えば特開平 10— 248929号、特開平 10— 3 14305 号などに開示されている。  The elastic body used for the protein prefilled syringe preparation of the present invention is usually produced by compression molding using the above rubber composition and a resin film. Such a stopper is disclosed in, for example, JP-A-10-248929 and JP-A-10-314305.
本発明において有効成分として使用するタンパク質は、例えば、顆粒球コロニ —刺激因子 （G— CSF) 、 顆粒球マクロファージコロニー刺激因子 (GM—C SF)、 エリスロポエチン (EP〇)、 トロンポポェチン等の造血因子、 イン夕一フ ェロン、 IL-1や IL-6等のサイト力イン、 モノクロ一ナル抗体、 組織プラスミノ —ゲン活性化因子 (TP A)、 ゥロキナーゼ、 血清アルブミン、 血液凝固第 V I I I因子、 レブチン、 インシュリン、 幹細胞成長因子 (S CF)などを含むが、 これ らに限定されない。 タンパク質の中でも、 EP〇、 G— CSF、 トロンボポェチ ン等の造血因子及びモノクロナール抗体が好ましく、さらに好ましくは E P〇、 G-C S F及びモノクローナル抗体である。  Proteins used as an active ingredient in the present invention include, for example, hematopoietic factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), erythropoietin (EP〇), and tropopoetin; Cytochromes such as IL-1 and IL-6, monoclonal antibodies, tissue plasminogen-activating factor (TPA), perokinase, serum albumin, blood coagulation factor VIII, lebutin, insulin , Including but not limited to, stem cell growth factor (SCF). Among proteins, hematopoietic factors such as EP〇, G-CSF, and thrombopoietin, and monoclonal antibodies are preferable, and EP〇, G-CSF and monoclonal antibodies are more preferable.
本発明において有効成分として使用するタンパク質とは、哺乳動物、特にヒト の生理活性タンパク質と実質的に同じ生物学的活性を有するものであり、天然由 来のもの、および遺伝子組換え法により得られたものを含むが、好ましいのは遺 伝子組換え法により得られたものである。特に、遺伝子組換え法で得られるタン パク質は、天然タンパク質に比べて不安定であり、本発明の製剤とすることが好 ましい。 また、遺伝子組換え法によって得られるタンパク質には天然タンパク質 とアミノ酸配列が同じであるもの、あるいは該アミノ酸配列の 1又は複数を欠失、 置換、 付加したもので前記生物学的活性を有するものを含む。 さらには、 タンパ ク質は P E G等により化学修飾されたものも含む。 The protein used as an active ingredient in the present invention is a protein having substantially the same biological activity as that of a biologically active protein of a mammal, particularly a human, and is obtained from a naturally-occurring protein or a recombinant gene. Preferred are those obtained by the gene recombination method. In particular, the protein obtained by the genetic recombination method is unstable compared to the natural protein, and it is preferable to use the preparation of the present invention. Good. Proteins obtained by the genetic recombination method include those having the same amino acid sequence as the natural protein, or those having one or more of the amino acid sequences deleted, substituted, or added and having the biological activity. Including. Furthermore, proteins include those chemically modified with PEG and the like.
本発明において有効成分として使用するタンパク質としては、例えば糖鎖を有 するタンパク質が挙げられる。 糖鎖の由来としては、 特に制限はないが、 哺乳動 物細胞に付加される糖鎖が好ましい。 哺乳動物細胞には、 例えば、 チャイニーズ ハムスター卵巣細胞 (CHO細胞)、 BHK細胞、 COS細胞、 ヒト由来の細胞等が あるが、 この中でも、 CHO細胞が最も好ましい。  Examples of the protein used as an active ingredient in the present invention include a protein having a sugar chain. The origin of the sugar chain is not particularly limited, but a sugar chain added to mammalian cells is preferable. Mammalian cells include, for example, Chinese hamster ovary cells (CHO cells), BHK cells, COS cells, human-derived cells and the like, among which CHO cells are most preferred.
本発明において有効成分として使用するタンパク質が EPOである場合には、 EP〇はいかなる方法で製造されたものでもよく、ヒト尿より種々の方法で抽出 し、 分離精製したもの、 遺伝子工学的手法 （例えば特開昭 61— 12288号） によりチャイニーズハムスター卵巣細胞 （CHO) 、 BHK細胞、 COS細胞、 ヒト由来の細胞などに産生せしめ、種々の方法で抽出し分離精製したものが用い られる。 さらには、 P EG等により化学修飾された EPOも含む （国際特許出願 公開番号 W090/12874参照） 。 さらに、 糖鎖のついていない E P Oを P EG等により化学修飾したものも含む。 また、 EPOのアミノ酸配列中の N—結 合炭水化物鎖結合部位もしくは O—結合炭水化物鎖結合部位において、 1以上の グリコシル化部位の数を増加させるように改変した EPO類似体も含む（例えば、 特開平 8— 151398号、 特表平 8— 506023号参照） 。 さらには、 糖鎖 結合部位の数は変化させずに、シアル酸等の含量を増加させることにより糖鎖の 量を増加させたものであってもよい。  When the protein used as the active ingredient in the present invention is EPO, EP〇 may be produced by any method, extracted from human urine by various methods, separated and purified, using a genetic engineering technique ( For example, according to Japanese Unexamined Patent Publication No. Sho 61-12288), hamster ovary cells (CHO), BHK cells, COS cells, human-derived cells, and the like, which are extracted and separated and purified by various methods, are used. Furthermore, it includes EPO chemically modified by PEG or the like (see International Patent Application Publication No. W090 / 12874). Furthermore, EPO with no sugar chain is chemically modified with PEG or the like. Also included are EPO analogs that have been modified to increase the number of one or more glycosylation sites at the N-linked carbohydrate chain binding site or the O-linked carbohydrate chain binding site in the amino acid sequence of EPO (eg, (See Kaihei 8—151398, Tokuhei 8-506023). Further, the amount of sugar chains may be increased by increasing the content of sialic acid or the like without changing the number of sugar chain binding sites.
本発明において有効成分として使用するタンパク質が G— C S Fである場合 には、 G— C S Fは高純度に精製された G— C S Fであれば全て使用できる。本 発明における G— C S Fは、 いかなる方法で製造されたものでもよく、 ヒト腫瘍 細胞の細胞株を培養し、 これから種々の方法で抽出し分離精製したもの、 あるい は遺伝子工学的手法により大腸菌などの細菌類；ィ一スト菌；チャイニーズハム スター卵巣 （CH〇） 細胞、 C 127細胞、 COS細胞などの動物由来の培養細 胞などに産生せしめ、種々の方法で抽出し分離精製したものが用いられる。好ま しくは大腸菌、ィースト菌又は CHO細胞によって遺伝子組換え法を用いて生産 されたものである。最も好ましくは CH〇細胞によって遺伝子組換え法を用いて 生産されたものである。 さらには、 P EG等により化学修飾された G— CSFも 含む （国際特許出願公開番号 W〇 90/ 12874参照） 。 When the protein used as an active ingredient in the present invention is G-CSF, any G-CSF can be used as long as it is highly purified G-CSF. The G-CSF in the present invention may be produced by any method, such as those obtained by culturing a cell line of human tumor cells and extracting and separating and purifying it from various cells, or E. coli by genetic engineering techniques. Bacteria; Listeria monocytogenes; Chinese hamster ovary (CH 、) cells, C127 cells, COS cells, and other animal-derived culture cells that are extracted and purified by various methods. Can be Like Alternatively, it is produced by E. coli, yeast, or CHO cells using a genetic recombination method. Most preferably, it is produced by a genetic modification method using CH 組 換 え cells. Furthermore, G-CSF chemically modified with PEG or the like is also included (see International Patent Application Publication No. WO 90/12874).
本発明において有効成分として使用する生理活性タンパク質がモノクロ一ナ ル抗体である場合には、モノクローナル抗体はいかなる方法で製造されたもので もよい。 モノクロ一ナ抗体は、 基本的には公知技術を使用し、 感作抗原を通常の 免疫方法にしたがって免疫し、得られる免疫細胞を通常の細胞融合法によって公 知の親細胞と融合させ、通常のスクリーニング法により、 モノクローナルな抗体 産生細胞をスクリーニングすることによって作成できる。 さらに、 モノクロ一ナ ル抗体は、ハイプリドーマが産生するモノクローナル抗体に限られるものではな く、ヒトに対する異種抗原性を低下させること等を目的として人為的に改変され たキメラ抗体を含む。 あるいは再構成（r e s h a p e d) したヒト型化抗体を 本発明に用いることもできる。 これはヒト以外の哺乳動物、たとえばマウス抗体 の相補性決定領域によりヒト抗体の相補性決定領域を置換したものであり、その 一般的な遺伝子組換手法も知られている。その既知方法を用いて、本発明に有用 な再構成ヒト型化抗体を得ることができる。  When the bioactive protein used as an active ingredient in the present invention is a monoclonal antibody, the monoclonal antibody may be produced by any method. Monoclonal antibodies are basically immunized with a sensitizing antigen according to a usual immunization method using a known technique, and the obtained immune cells are fused with a known parent cell by a normal cell fusion method. Can be prepared by screening monoclonal antibody-producing cells by the screening method described above. Further, the monoclonal antibody is not limited to the monoclonal antibody produced by the hybridoma, and includes a chimeric antibody artificially modified for the purpose of, for example, reducing the xenoantigenicity to humans. Alternatively, a reshaped (reshaped) humanized antibody can also be used in the present invention. This is obtained by replacing the complementarity-determining region of a human antibody with the complementarity-determining region of a non-human mammal, for example, a mouse antibody, and a general gene recombination technique is also known. Using the known method, a reshaped humanized antibody useful in the present invention can be obtained.
なお、必要に応じ、再構成ヒト型化抗体の相補性決定領域が適切な抗原結合部 位を形成するように抗体の可変領域のフレームワーク （FR)領域のアミノ酸を 置換してもよい （S a t oら、 C anc e r Re s. 53 : 1— 6， 1993) 。 このような再構成ヒト型化抗体として、抗 I L一 6レセプ夕一ヒト型化抗体であ るヒト型化 PM— 1 (hPM— 1)抗体が好ましく例示される （国際特許出願公 開番号 WO 92 - 19759を参照） 。  If necessary, amino acids in the framework (FR) region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped humanized antibody forms an appropriate antigen-binding site (S ato et al., Cancer Res. 53: 1-6, 1993). A preferred example of such a reshaped humanized antibody is a humanized PM-1 (hPM-1) antibody which is an anti-IL-16 receptor humanized antibody (International Patent Application Publication No. WO 92-19759).
さらに、本発明の抗体としては、 トランスジエニック動物等を使用して周知の 技術により製造されたヒト抗体も含まれる。  Furthermore, the antibodies of the present invention also include human antibodies produced by known techniques using transgenic animals and the like.
本発明は、 抗体断片 （例えば、 F ab、 F (a b) 2など） や改変抗体 （例え ば、 一本鎖抗体など） にも適用できる。 The present invention can be applied to antibody fragments (for example, Fab, F (ab) 2, etc.) and modified antibodies (for example, single-chain antibodies, etc.).
特に、 キメラ抗体、 ヒト型化抗体、 抗体断片及び改変抗体は、 天然の抗体に比 ベて不安定であるため、 本発明の製剤を使用することが好ましい。 本発明のタンパク質製剤には、 希釈剤、 溶解補助剤、 等張化剤、 賦形剤、 p H 調整剤、 無痛化剤、 緩衝剤、 含硫還元剤、 酸化防止剤等を含有してもよい。 例え ば、 等張化剤としては、 ポリエチレングリコール；デキストラン、 マンニトール、 ソルビト一ル、 イノシトール、 グルコース、 フラク 1 ^一ス、 ラク！ ^一ス、 キシロ ース、 マンノース、 マル! ス、 シユークロース、 ラフイノースなどの糖類を用 いることができる。 含硫還元剤としては、 N—ァセチルシスティン、 N—ァセチ ルホモシスティン、 チォクト酸、 チォジグリコール、 チォエタノールァミン、 チ オダリセロール、 チォソルビトール、 チォグリコール酸およびその塩、 チォ硫酸 ナトリゥム、 ダル夕チオン、並びに炭素原子数 1〜 7のチオアルカン酸等のスル フヒドリル基を有するもの等が挙げられる。 また、 酸化防止剤としては、 エリソ ルビン酸、 ジブチルヒドロキシトルエン、 ブチルヒドロキシァニソ一ル、 α—ト コフエロール、 酢酸トコフエロール、 L—ァスコルビン酸およびその塩、 L—ァ スコルビン酸パルミテート、 L—ァスコルビン酸ステアレート、亜硫酸水素ナト リウム、 亜硫酸ナトリウム、 没食子酸トリアミル、 没食子酸プロピルあるいはェ チレンジァミン四酢酸ニナトリウム （E D T A) 、 ピロリン酸ナトリウム、 メタ リン酸ナトリウム等のキレ一ト剤が挙げられる。 さらには、 塩化ナトリウム、 塩 化カリウム、 塩化カルシウム、 リン酸ナトリウム、 リン酸カリウム、 炭酸水素ナ トリウムなどの無機塩；クェン酸ナトリウム、 クェン酸カリウム、 酢酸ナトリウ ムなどの有機塩などの溶液製剤に通常添加される成分を含んでいてもよい。 In particular, chimeric antibodies, humanized antibodies, antibody fragments, and modified antibodies are more unstable than natural antibodies, and therefore it is preferable to use the formulations of the present invention. The protein preparation of the present invention may contain a diluent, a solubilizing agent, a tonicity agent, an excipient, a pH adjuster, a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, etc. Good. For example, as the tonicity agent, polyethylene glycol; dextran, mannitol, sorbitol, inositol, glucose, flak, and lac! You can use sugars such as sucrose, xylose, mannose, maltose, sucrose, and raffinose. Examples of the sulfur-containing reducing agent include N-acetyl cysteine, N-acetyl homocystine, thioctic acid, thiodiglycol, thioethanolamine, thiodaricerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, and dal. And those having a sulfhydryl group such as thiothione and thioalkanoic acid having 1 to 7 carbon atoms. Examples of antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, and L-ascorbic acid Chelating agents such as stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or disodium ethyl diamine tetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate and the like. In addition, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium hydrogen carbonate; and solution preparations such as organic salts such as sodium citrate, potassium citrate, and sodium acetate. It may contain components that are usually added.
本発明のタンパク質溶液製剤にはさらに、各タンパク質に適切な安定化剤を含 んでいてもよく、 安定化剤には界面活性剤 （例えば、 非イオン界面活性剤である ソルビタン脂肪酸エステル、 グリセリン脂肪酸エステル、ポリグリセリン脂肪酸 エステル、 ポリオキシエチレンソルビ夕ン脂肪酸エステル、ポリオキシエチレン ソルビット脂肪酸エステル、 ポリオキシエチレングリセリン脂肪酸エステル、 ポ リエチレングリコール脂肪酸エステル、ポリオキシエチレンアルキルエーテル、 ポリオキシエチレンポリオキシプロピレンアルキルエーテル、ポリオキシェチレ ンアルキルフエニルエーテル、 ポリオキシエチレン硬化ヒマシ油、 ポリオキシェ チレンミツロウ誘導体、 ポリオキシエチレンラノリン誘導体、ポリオキシェチレ ン脂肪酸アミド；陰イオン界面活性剤であるアルキル硫酸塩、 ポリオ： ンアルキルエーテル硫酸塩、 アルキルスルホコハク酸エステル塩；天然系の界面 活性剤であるレシチン、 グリセ口リン脂質、 スフインゴリン脂質、 ショ糖脂肪酸 エステルなどが挙げられ、特にポリオキシエチレンソルビタン脂肪酸エステルが 好ましく、 とりわけポリオキシエチレンソルビ夕ンモノォレエ一ト （ポリソルべ —ト 80)およびポリオキシエチレンソルビタンモノラウレ一ト （ポリソルべ一 ト 20) が好ましい） 、 およびアミノ酸 （例えば、 D―、 L—および DL—体の ロイシン、 トリプトファン、 セリン、 グルタミン酸、 アルギニン、 ヒスチジン、 リジン、 メチォニン、 フエ二ルァラニンおよびァセチルトリブトファンならびに その塩であり、 より好ましいのは L—ロイシン、 L—トリブトファン、 L—ダル 夕ミン酸、 L—アルギニン、 L—ヒスチジンおよび L—リジンならびにその塩で ある） などを含むが、 これに限定されない。 The protein solution preparation of the present invention may further contain an appropriate stabilizer for each protein, and the stabilizer includes a surfactant (for example, sorbitan fatty acid ester or glycerin fatty acid ester which is a nonionic surfactant). , Polyglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbite fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene alkyl ether, Polyoxyethylene alkylphenyl ether, polyoxyethylene hydrogenated castor oil, polyoxyethylene beeswax derivative, polyoxyethylene lanolin derivative, polyoxyethylene fatty acid Amide; alkyl sulfate as anionic surfactant, polio: Alkyl ether sulfates and alkyl sulfosuccinates; natural surfactants such as lecithin, glycerol phospholipids, sphingophospholipids, and sucrose fatty acid esters, and particularly preferred are polyoxyethylene sorbitan fatty acid esters, Polyoxyethylene sorbitan monooleate (polysorbate 80) and polyoxyethylene sorbitan monolaurate (polysorbate 20) are preferred, and amino acids (eg, D-, L- and DL-forms) Of leucine, tryptophan, serine, glutamic acid, arginine, histidine, lysine, methionine, fenylalanine and acetyltributophane and salts thereof, and more preferred are L-leucine, L-tributophan, and L-dalminic acid. , L Arginine, L- histidine and a L- lysine, and their salts) including such as, but not limited thereto.
本発明の安定なタンパク質プレフィルドシリンジ製剤は通常非経口投与経路 で、 例えば注射剤 （皮下又は静注） 、 経皮、 経粘膜、 経鼻などで投与されるが、 経口投与も可能である。  The stable protein prefilled syringe preparation of the present invention is usually administered by parenteral administration route, for example, injection (subcutaneous or intravenous injection), transdermal, transmucosal, nasal, etc., but oral administration is also possible.
本発明の安定なタンパク質プレフィルドシリンジ製剤中に含まれる夕ンパク 質の量は、 使用するタンパク質、 治療すべき疾患の種類、 疾患の重症度、 患者の 年齢などに応じて決定できる。  The amount of protein contained in the stable protein prefilled syringe formulation of the present invention can be determined according to the protein used, the type of disease to be treated, the severity of the disease, the age of the patient, and the like.
一般的には、 0. 01 g/m 1以上、 好ましくは 0. 1 gZm 1以上、 さ らに好ましくは 1 g Zm 1以上、 さらに好ましくは 10 /i g Zm 1以上、 さら に好ましくは 50 g/m 1以上のタンパク質を含む。特に、 プレフィルドシリ ンジ溶液製剤の場合には、 0. 1 gZml以上、 好ましくは 1 /x gノ m l以上、 さらに好ましくは 10 g /m 1以上、さらに好ましくは 50 g Zm 1以上の タンパク質を含む。  Generally, 0.01 g / m1 or more, preferably 0.1 gZm1 or more, more preferably 1 gZm1 or more, more preferably 10 / ig Zm1 or more, further preferably 50 g / m Contains 1 or more proteins. In particular, in the case of a prefilled syringe solution preparation, it contains 0.1 gZml or more, preferably 1 / xg noml or more, more preferably 10 g / m1 or more, and still more preferably 50 gZm1 or more.
例えば EPOの場合には溶液製剤中の EP〇の量は、一般には 100〜500 000 I UZmし好ましくは 200〜 100000 I UZmし さらに好まし くは 750〜 72000 1 U/m 1である。 また、 G— C S Fの場合には一般に は l〜1000 g /m 1、好ましくは 10〜800 //g Zmし さらに好まし くは 50〜 500 /i g/m 1である。 また、抗 I L一 6レセプ夕一ヒト型化抗体 の場合には、 一般には最終投与濃度で 0. 1〜20 OmgZmし 好ましくは 1 〜 12 Omg/m 1である。 For example, in the case of EPO, the amount of EP〇 in the solution formulation is generally between 100 and 500 000 I UZm, preferably between 200 and 100000 I UZm and more preferably between 750 and 72000 1 U / m 1. In the case of G-CSF, it is generally l to 1000 g / m 1, preferably 10 to 800 // g Zm, and more preferably 50 to 500 / ig / m 1. In addition, in the case of an anti-IL-16 receptor humanized antibody, the final administration concentration is generally 0.1 to 20 OmgZm, preferably 1 to 20 OmgZm. ~ 12 Omg / m1.
本発明のタンパク質プレフィルドシリンジ製剤はこれらの成分をリン酸およ び Z又はクェン酸緩衝液などの溶液製剤の分野で公知の水性緩衝液に溶解する ことによって調製できる。 リン酸緩衝液は、 リン酸一水素ナトリウム一リン酸二 水素ナトリゥム系が好ましく、クェン酸緩衝液としてはクェン酸ナトリウムの緩 衝液が好ましい。 さらに、 タンパク質の種類によっては、 グリシン緩衝液やヒス チジン緩衝液などの緩衝液を用いてもよい。  The protein prefilled syringe formulation of the present invention can be prepared by dissolving these components in an aqueous buffer known in the field of solution formulation, such as phosphate and Z or citrate buffers. The phosphate buffer is preferably a sodium monohydrogen phosphate sodium dihydrogen phosphate system, and the citrate buffer is preferably a sodium citrate buffer solution. Further, depending on the type of protein, a buffer such as a glycine buffer or a histidine buffer may be used.
本発明の夕ンパク質プレフィルドシリンジ製剤がェリス口ポェチン溶液製剤 である場合には、 その中には EP〇、 非イオン性界面活性剤 （例えばポリソルべ ート 80、 ポリソルベート 20) 、 等張剤 （例えば塩化ナトリウム） および必要 に応じて安定化剤 （例えばアミノ酸、 好ましくは L一ヒスチジン） を含み、 pH を 5. 0— 8. 0、 好ましくは 5. 5- 7. 0とすることが好ましい。  When the evening protein prefilled syringe preparation of the present invention is a erythrocyte poetin solution preparation, it contains EP〇, a nonionic surfactant (for example, polysorbate 80, polysorbate 20), an isotonic agent ( It preferably contains, for example, sodium chloride) and, if necessary, a stabilizer (eg, an amino acid, preferably L-histidine), and has a pH of 5.0 to 8.0, preferably 5.5 to 7.0.
本発明のタンパク質プレフィルドシリンジ製剤が G— C S F溶液製剤である 場合には、 その中には G— CSF、 非イオン性界面活性剤 （例えばポリソルベー ト 80、 ポリソルベート 20) 、 および必要に応じて希釈剤、 溶解補助剤、 等張 化剤、 賦形剤、 pH調整剤、 無痛化剤、 緩衝剤、 含硫還元剤、 酸化防止剤等を含 み、 pHを 5. 0- 7. 0、 好ましくは 6— 6. 8とすることが好ましい。 本発明のタンパク質プレフィルドシリンジ製剤が抗 I L一 6レセプ夕ーヒト 型化抗体溶液製剤である場合には、抗 I L一 6レセプ夕一ヒト型化抗体を好まし くはグリシン緩衝液及び Z又はヒスチジン緩衝液などの緩衝液に溶解し、その他 に非イオン性界面活性剤 （例えばポリソルベート 80、 ポリソルベート 20 )、 および必要に応じて希釈剤、 溶解補助剤、 等張化剤、 賦形剤、 pH調整剤、 無痛 化剤、 緩衝剤、 含硫還元剤、 酸化防止剤等を含み、 pHを 5— 8とすることが好 ましい。  When the protein prefilled syringe preparation of the present invention is a G-CSF solution preparation, the preparation contains G-CSF, a nonionic surfactant (for example, polysorbate 80, polysorbate 20), and if necessary, a diluent. , A solubilizing agent, a tonicity agent, an excipient, a pH adjuster, a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, etc., and a pH of 5.0-7.0, preferably It is preferred to be 6-6.8. When the protein prefilled syringe preparation of the present invention is an anti-IL16 receptor-humanized antibody solution preparation, the anti-IL16 receptor humanized antibody is preferably used in a glycine buffer and a Z or histidine buffer. Dissolved in a buffer solution such as a liquid, and other nonionic surfactants (for example, polysorbate 80, polysorbate 20), and if necessary, diluents, dissolution aids, isotonic agents, excipients, and pH adjusters. It preferably contains a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, and the like, and has a pH of 5 to 8.
本発明のプラスチックフィルムを少なくともタンパク質溶液接触面にラミネ 一トしたストッパーを具備した注射器に予め充填された安定なタンパク質プレ フィルドシリンジ製剤は G— C S Fを用いて試験した後述する実施例に示すよ うに、 40°C— 2週間の加速試験を行った後にも、 ラミネートしないストッパー に比較して極めて良好な G— CSF残存率を示す。 あるいは 25°Cで 6ヶ月、 1 0°Cで 1年という長期間保存した後にも、 良好な G— CSF残存率を示す。 また、 タンパク質が EPOの場合にも、 50°C— 2週間もしくは 40°C— 1ヶ月の加速 試験を行った後にも、 あるいは 25°Cで 6ヶ月、 1 0°Cで 1年の長期間保存した 後にも、 極めて良好な EPO残存率を示す。 A stable protein pre-filled syringe formulation pre-filled in a syringe equipped with a stopper in which the plastic film of the present invention was laminated at least on the protein solution contact surface was tested using G-CSF, as shown in Examples described later. Even after a 2-week accelerated test at 40 ° C, the G-CSF retention rate is much better than that of a non-laminated stopper. Or 6 months at 25 ° C, 1 Even after long-term storage at 0 ° C for 1 year, it shows good G-CSF retention. Also, if the protein is EPO, even after accelerated tests at 50 ° C-2 weeks or 40 ° C-1 month, or at 6 months at 25 ° C and 1 year at 10 ° C Very good EPO persistence after storage.
本発明はまた、プラスチックフィルムを少なくともタンパク質溶液接触面にラ ミネートしたストッパーを具備した注射器にタンパク質溶液製剤を充填して保 存することからなるタンパク質プレフィルドシリンジ製剤の安定化方法を提供 する。本発明における安定化とは、充填するタンパク質の種類によって異なるが、 例えば G— CSF溶液製剤（G— CSFを 12 含有） の場合であれ ば、 これを例えば 40°Cで 2週間以上保存し、その際に G— CS Fの残存率を 7 5%以上、好ましくは 80%以上、 さらに好ましくは 85%以上に保つことを意 味する。 あるいは、 G— CSF溶液製剤 （G— CS Fを 250 /x g/m l含有） を 25 °Cで 6ヶ月保存した後に、 G— CSFの残存率を 80 %以上、好ましくは 85%以上、 さらに好ましくは 90 %以上に保つことを意味する。  The present invention also provides a method for stabilizing a protein prefilled syringe formulation, which comprises filling and storing a protein solution formulation in a syringe provided with a stopper having a plastic film laminated on at least the protein solution contact surface. The stabilization in the present invention depends on the type of protein to be filled. For example, in the case of a G-CSF solution preparation (containing 12 G-CSF), this is stored at, for example, 40 ° C for 2 weeks or more. At that time, it means that the residual ratio of G-CSF is maintained at 75% or more, preferably 80% or more, and more preferably 85% or more. Alternatively, after storing a G-CSF solution preparation (containing 250 g / ml of G-CSF) at 25 ° C for 6 months, the residual ratio of G-CSF is 80% or more, preferably 85% or more, more preferably Means to keep it above 90%.
本発明によってタンパク質プレフィルドシリンジ製剤を常温で安定に長期保 存することが可能となる。 産業上の利用可能性  According to the present invention, a protein prefilled syringe preparation can be stably stored at room temperature for a long period of time. Industrial applicability
本発明のプラスチックフィルムを少なくともタンパク質溶液接触面にラミネ 一卜したストッパーを具備した、安定したタンパク質プレフィルドシリンジ製剤 は、 タンパク質の生理活性含量が長期にわたって低下せず、従来のゴムストッパ 一を使用したプレフィルドシリンジ製剤よりも安定である。本発明により、 タン パク質プレフィルドシリンジ製剤を常温で長期間安定に保存することが可能に なる。 特に、 タンパク質が遺伝子組換え法で得られるものである場合には、 天然 夕ンパク質に比べて不安定であり、本発明のプレフィルドシリンジ製剤とするこ とにより常温で長期間安定に保存することができる。 実施例  A stable protein prefilled syringe formulation comprising a stopper in which the plastic film of the present invention is laminated at least on the protein solution contacting surface is a stable protein prefilled syringe formulation. More stable than syringe formulations. According to the present invention, a protein prefilled syringe preparation can be stably stored at room temperature for a long period of time. In particular, when the protein is obtained by a genetic recombination method, it is unstable compared to natural proteins, and should be stably stored at room temperature for a long period of time by using the prefilled syringe preparation of the present invention. Can be. Example
以下の実施例では、 顆粒球コロニー刺激因子（G— CSF) を代表例として用 いて、加速試験を実施した結果を記載するが、本発明の範囲はこれに限定されな レ^ 種々の変更、 修飾が当業者には可能である。 In the following examples, granulocyte colony stimulating factor (G-CSF) is used as a representative example. The results of an accelerated test are described below, but the scope of the present invention is not limited to this. Various changes and modifications can be made by those skilled in the art.
なお、以下の実施例において、製剤の評価は RP—HPLC分析法を用いて G -C S Fの含量を求めることにより行った。  In the following examples, the evaluation of the preparation was performed by determining the content of G-CSF using RP-HPLC analysis.
実施例 1 ： G— CS F溶液製剤の 40°C— 2週間加速試験 Example 1: G-CSF solution at 40 ° C for 2 weeks accelerated test
G— CS F溶液製剤の調製 Preparation of G-CSF solution preparation
調剤溶液 1 m 1中に以下の成分：  The following ingredients in 1 ml of the preparation solution:
G-C S F 1 25 g (ポリソルべ一ト 20) 0. lmg  G-C S F 1 25 g (Polysorbate 20) 0.lmg
塩化ナトリウム 7. 5mg  Sodium chloride 7.5mg
を含み、 lmo 1 ZL塩酸にて pH6. 5に調整した。 And adjusted to pH 6.5 with 1mo 1 ZL hydrochloric acid.
試験方法 Test method
前記のようにして調製した G— CS F溶液製剤を無菌濾過した後、 0. 5mL ずつを下記表 1に示す容器形態に無菌的に充填し、 打栓を施した。  After the G-CSF solution preparation prepared as described above was aseptically filtered, 0.5 mL each was aseptically filled into the container form shown in Table 1 below and stoppered.
なお、本実施例で使用した G— CS Fは CHO細胞で産生された糖鎖を有する 組換えタンパク質 （rG-CSF) である。 容器形態  The G-CSF used in this example is a recombinant protein having a sugar chain (rG-CSF) produced in CHO cells. Container form
PTFEラミネート ガラスシリンジに上記調剤液を充填し、 WEST社製スト ストッパー ッパ一 （材質：ブロモブチルゴム PH4023/50) の接液 面に PTFE シ一トを貼り付けたもので密封打栓したも の。  A PTFE-laminated glass syringe is filled with the above dispensing solution, and the stopper is sealed and stoppered by attaching a PTFE sheet to the liquid contacting surface of a WEST stop stopper (Material: bromobutyl rubber PH4023 / 50). .
未処理ストッパー ガラスシリンジに上記調剤液を充填し、 WEST社製スト ッパー （材質：ブロモブチルゴム PH4023/50) で密封 打栓したもの。 Untreated stopper A syringe filled with the above preparation liquid and sealed with a stopper (material: bromobutyl rubber PH4023 / 50) manufactured by WEST.
PTFE ラミネ一トストツ あらかじめ WEST社製ストツパ一 （材質： ブロモブチ パーから PTFE を剥離さ ルゴム PH4023/50)の接液面に PTFEシ一トを貼り付け せたもの たものから、 接液面の PTFE シートを剥離させた。 ガ ラスシリンジに上記調剤液を充填し， 本ストッパーを 使用して密封打栓したもの。  PTFE laminating stock A PTFE sheet with a PTFE sheet attached to the liquid contacting surface of a WEST stopper (material: PTFE is peeled from bromobutyper), and the liquid contacting surface is used. Was peeled off. A glass syringe filled with the above dispensing solution and sealed and stoppered using this stopper.
ガラスバイアル 未処理白色ガラスバイアルに上記調剤液を充填し， 大 協精ェ社製ゴム栓 S5- F3で打栓したもの。 このように， 無菌的に調製 ·濾過を行い製造した表 1記載の G-CSF調剤液を含 む各容器形態は， 40°Cの恒温槽において 2週間の加速に供された。未加速品試料， および， 40°C- 2 週間加速品試料について， C4 逆相カラム （4. 6iMi X 250imn， 300 オングストローム） を用い， 純水， ァセトニトリル， トリフルォロ酢酸を移動相 に用いた逆相系高速液体クロマトグラフィー法により G- CSF含量を測定した。 Glass vial An untreated white glass vial filled with the above preparation liquid and stoppered with a rubber stopper S5-F3 manufactured by Daikyo Seiki. Each container form containing the G-CSF preparations shown in Table 1 aseptically prepared and filtered was subjected to two-week acceleration in a constant-temperature bath at 40 ° C. For the unaccelerated sample and the sample accelerated for 2 weeks at 40 ° C, reverse phase using pure water, acetonitrile, and trifluoroacetic acid as the mobile phase using a C4 reversed-phase column (4.6 iMi X 250 imn, 300 Å). G-CSF content was measured by high-performance liquid chromatography.
G - CSFとして， 相当量を注入し， ァセトニトリルのグラジェントにより G- CSFを溶出させ， 215ηπιの波長で分光学的に検出し， G- CSF含量を測定した。 本方法で測定した G- CSF含量を用い， 下記の式に基づき 40°C- 2週間加速後の 残存率 （％) を算出した。 A considerable amount of G-CSF was injected, and G-CSF was eluted with an acetonitrile gradient, and the G-CSF content was measured by spectroscopic detection at a wavelength of 215ηπι. Using the G-CSF content measured by this method, the survival rate (%) after acceleration at 40 ° C for 2 weeks was calculated based on the following equation.
(40°C-2週間加速品試料の G- CSF含量） (G-CSF content of accelerated sample at 40 ° C for 2 weeks)
残存率 （％) = X 100  Survival rate (%) = X 100
(未加速品試料の G-CSF含量）  (G-CSF content of unaccelerated sample)
得られた結果を以下の表 2に示す The results obtained are shown in Table 2 below.
表 2 Table 2
Figure imgf000015_0001
表 2に示すとおり、未処理ストッパーを使用したときに認められる G- CSFの不 安定化は， PTFE ラミネートストッパー使用品においては全く認められず、 ガラ スパイアル使用品と同等の良好な安定性を示した。 また、 PTFE シートを剥離さ せた PTFEラミネートストッパーを使用した場合には， 未処理ストッパー使用品 と同程度の不安定化を認めたことから， PTFE ラミネートの重要性が明確に示さ れた。 実施例 2 ： G-CSF溶液製剤の 25°C及び 10°Cの保存安定性試験
Figure imgf000015_0001
As shown in Table 2, the instability of G-CSF observed when using the untreated stopper was not observed at all with the product using the PTFE laminate stopper, and showed the same good stability as the product using the glass spear. Was. In addition, when a PTFE laminate stopper with a PTFE sheet peeled was used, the same degree of instability as the untreated stopper was used, indicating the importance of the PTFE laminate. Example 2: Storage stability test of G-CSF solution formulation at 25 ° C and 10 ° C
実施例 1で用いた G— C S F溶液製剤 （1 2 5 /x gZlm l) に加えて、 以下 の高 DOSE製剤を調製した。  In addition to the G—CSF solution preparation (125 / xgZlm) used in Example 1, the following high DOSE preparation was prepared.
G-CS F (2 5 0 /2 R/lm 1 ) 溶液製剤の調製  Preparation of G-CS F (250/2 R / lm 1) solution preparation
調剤溶液 1 m 1中に以下の成分：  The following ingredients in 1 ml of the preparation solution:
G-C S F 2 5 0 g ポ 卜 GC SF 250 g Port
(ポリソルべ一卜 20) 0 1 mg  (Polysorbate 20) 0 1 mg
塩化ナトリウム 6 Omg  Sodium chloride 6 Omg
を含み、 lmo 1 /L塩酸にて pH 6. 5に調整した, And adjusted to pH 6.5 with lmo 1 / L hydrochloric acid,
試験方法 Test method
記のようにして調製した 2種の G— CS F溶液製剤を無菌濾過した後、 0  After sterile filtration of the two G—CSF solution preparations prepared as described above,
5mLずつを PTFEラミネートストッパー （A〜E) を備えた容器に無菌的に充填 し、 打栓を施した。  5 mL each was aseptically filled into a container equipped with a PTFE laminate stopper (A to E) and stoppered.
このように， 無菌的に調製 ·濾過を行い製造した溶液製剤を含む各容器形態は， 25°C及び 1 0°Cで長期保存した。その後、実施例 1と同様に残存率を算出した。 結果を表 3、 表 4に示す。  In this way, each container form containing the solution preparation prepared by aseptic preparation and filtration was stored at 25 ° C and 10 ° C for a long time. Thereafter, the residual ratio was calculated in the same manner as in Example 1. Tables 3 and 4 show the results.
表 3 Table 3
125/z /mL (0.5mL充填）  125 / z / mL (0.5mL filling)
容器形態 25°Cでの残存率 (％) 10°Cでの残存率 (％)  Container form Residual rate at 25 ° C (%) Residual rate at 10 ° C (%)
2力月 4力月 6力月 3力月 6力月 1年 2 months 4 months 6 months 3 months 6 months 1 year
PTFEラミネートス卜ッパー， 92.6 90.1 85.4 94.7 93.7 90.7 APTFE laminated stripper, 92.6 90.1 85.4 94.7 93.7 90.7 A
PTFEラミネートストツパー， 88.9 85.8 72.9 91.9 93.1 85.5 BPTFE laminated stopper, 88.9 85.8 72.9 91.9 93.1 85.5 B
PTFEラミネートストッパー， 91.2 90.2 78.1 94.1 93.4 89.9 CPTFE laminated stopper, 91.2 90.2 78.1 94.1 93.4 89.9 C
PTFEラミネートストツパー， 92.3 89.8 77.9 94.8 92.8 89.6 DPTFE laminated stopper, 92.3 89.8 77.9 94.8 92.8 89.6 D
PTFEラミネ一トストツパー， 91.6 89.8 84.0 94.2 92.7 88.4 E PTFE laminating stopper, 91.6 89.8 84.0 94.2 92.7 88.4 E
表 4 Table 4
Figure imgf000017_0001
表から明らかなように、 界面活性剤、塩化ナトリウム以外に安定化剤を含んで いない G— C S F製剤においても、 良好な残存率を示した。 実施例 3 ： EPO溶液製剤の安定性試験
Figure imgf000017_0001
As is clear from the table, the G-CSF preparation containing no stabilizer other than the surfactant and sodium chloride exhibited a good residual ratio. Example 3: Stability test of EPO solution preparation
以下の 2種の濃度 （1500IUZmL及び 48000IU/mL) の E P O溶液製剤を 調製し、 PTFEラミネートストッパー （B〜D) を備えた容器に無菌的に充填し、 打栓を施した。 5 0 °Cと 4 0 °Cで加速試験を行い、 また 2 5 °Cと 1 0 °Cで長期保 存試験を行い、 それぞれ残存率を算出した。 結果を表 5— 8に示す。 処方： 1500IU L 48000IU/mL  EPO solution preparations of the following two concentrations (1500 IUZmL and 48000 IU / mL) were prepared, filled aseptically into containers equipped with PTFE laminate stoppers (BD), and stoppered. Accelerated tests were performed at 50 ° C and 40 ° C, and long-term storage tests were performed at 25 ° C and 10 ° C, and the residual rates were calculated respectively. The results are shown in Table 5-8. Formulation: 1500IU L 48000IU / mL
EPOCH 1500IU 48000IU ポリソルベート 80 0.05 mg 0.05 mg L-塩酸ヒスチジン 1.35 mg 1.35 mg 塩化ナトリウム 7.74 mg 2.26 mg リン酸緩衝液 10mM (pH6.0) 25mM(pH6.0) 表 5
Figure imgf000018_0002
表 6
Figure imgf000018_0003
表 7
Figure imgf000018_0001
8 EPOCH 1500IU 48000IU Polysorbate 80 0.05 mg 0.05 mg L-Histidine hydrochloride 1.35 mg 1.35 mg Sodium chloride 7.74 mg 2.26 mg Phosphate buffer 10 mM (pH 6.0) 25 mM (pH 6.0) Table 5
Figure imgf000018_0002
Table 6
Figure imgf000018_0003
Table 7
Figure imgf000018_0001
8
Figure imgf000019_0001
いずれの溶液製剤も極めて良好な残存率を示した。
Figure imgf000019_0001
All solution preparations showed extremely good residual rates.

Claims

請求の範囲 The scope of the claims
1 . 少なくともタンパク質溶液接触面に樹脂を被覆した弾性体を具備した、安 定したタンパク質プレフィルドシリンジ製剤。 1. A stable protein prefilled syringe formulation comprising an elastic body coated with a resin at least on the protein solution contact surface.
2 . 樹脂フィルムを少なくともタンパク質溶液接触面にラミネートしたストツ パーを具備した、請求項 1記載の安定したタンパク質プレフィルドシリンジ製剤。2. The stable protein prefilled syringe preparation according to claim 1, further comprising a stopper having a resin film laminated on at least a protein solution contact surface.
3 . 樹脂がポリエチレン、 ポリプロピレン、 ポリカーボネート、 ポリエステル、 フッ素系樹脂から選択される請求項 1又は 2記載のタンパク質プレフィルドシ リンジ製剤。 3. The protein prefilled syringe preparation according to claim 1, wherein the resin is selected from polyethylene, polypropylene, polycarbonate, polyester, and fluororesin.
4 . 樹脂がフッ素系樹脂である請求項 3記載の夕ンパク質プレフィルドシリン ジ製剤。 4. The preprotein syringe preparation according to claim 3, wherein the resin is a fluororesin.
5 . 樹脂がポリテトラフルォロエチレンである請求項 4記載のタンパク質プレ フィルドシリンジ製剤。  5. The protein prefilled syringe preparation according to claim 4, wherein the resin is polytetrafluoroethylene.
6 . タンパク質が遺伝子組換えタンパク質である請求項 1〜 5のいずれかに記 載のタンパク質プレフィルドシリンジ製剤。  6. The protein prefilled syringe preparation according to any one of claims 1 to 5, wherein the protein is a recombinant protein.
7 . タンパク質がエリスロポエチンである請求項 6記載のタンパク質プレフィ ルドシリンジ製剤。  7. The protein pre-filled syringe preparation according to claim 6, wherein the protein is erythropoietin.
8 . タンパク質が顆粒球コロニー刺激因子である請求項 6記載のタンパク質プ レフイリレドシリンジ製剤。  8. The protein preeryred syringe preparation according to claim 6, wherein the protein is a granulocyte colony stimulating factor.
9 . タンパク質が糖鎖を有するタンパク質である請求項 1〜8のいずれかに記 載のタンパク質プレフィルドシリンジ製剤。  9. The protein prefilled syringe preparation according to any one of claims 1 to 8, wherein the protein is a protein having a sugar chain.
1 0 . 少なくともタンパク質溶液接触面に樹脂を被覆した弾性体を具備した注 射器にタンパク質溶液製剤を充填して保存することからなるタンパク質プレフ ィルドシリンジ製剤の安定化方法。  10. A method for stabilizing a protein prefilled syringe formulation, which comprises filling and storing a protein solution formulation in an injector having an elastic body coated with a resin on at least the protein solution contact surface.
PCT/JP2000/009309 1999-12-28 2000-12-27 Protein-prefilled syringe preparation and method of stabilizing the same WO2001047544A1 (en)

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JP2012228335A (en) * 2011-04-26 2012-11-22 Taisei Kako Co Ltd Elastic sealing body for prefilled syringe

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Publication number Priority date Publication date Assignee Title
JPS5219435Y2 (en) * 1971-06-11 1977-05-04
JPS5532602Y2 (en) * 1972-05-31 1980-08-04
JPS62139668A (en) * 1985-12-16 1987-06-23 株式会社大協精工 Laminated plug for syringe
EP0264273A2 (en) * 1986-10-15 1988-04-20 Daikyo Gomu Seiko Ltd. A laminated sliding stopper for a syringe
EP0879611A2 (en) * 1997-05-22 1998-11-25 Daikyo Seiko, Ltd. A sealing stopper for a syringe and a prefilled syringe
JPH11146910A (en) * 1997-04-25 1999-06-02 Minoru Terano Biocompatible polyolefin molding and biocompatible polymeric material

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Publication number Priority date Publication date Assignee Title
JPS5219435Y2 (en) * 1971-06-11 1977-05-04
JPS5532602Y2 (en) * 1972-05-31 1980-08-04
JPS62139668A (en) * 1985-12-16 1987-06-23 株式会社大協精工 Laminated plug for syringe
EP0264273A2 (en) * 1986-10-15 1988-04-20 Daikyo Gomu Seiko Ltd. A laminated sliding stopper for a syringe
JPH11146910A (en) * 1997-04-25 1999-06-02 Minoru Terano Biocompatible polyolefin molding and biocompatible polymeric material
EP0879611A2 (en) * 1997-05-22 1998-11-25 Daikyo Seiko, Ltd. A sealing stopper for a syringe and a prefilled syringe

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012228335A (en) * 2011-04-26 2012-11-22 Taisei Kako Co Ltd Elastic sealing body for prefilled syringe
EP2703024A1 (en) * 2011-04-26 2014-03-05 Taisei Kako Co., Ltd. Elastic sealing body for prefilled syringe
EP2703024A4 (en) * 2011-04-26 2014-10-22 Taisei Kako Co Elastic sealing body for prefilled syringe

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