WO1999018119A1 - Procede de lyophilisation de proteines - Google Patents

Procede de lyophilisation de proteines Download PDF

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Publication number
WO1999018119A1
WO1999018119A1 PCT/JP1998/004416 JP9804416W WO9918119A1 WO 1999018119 A1 WO1999018119 A1 WO 1999018119A1 JP 9804416 W JP9804416 W JP 9804416W WO 9918119 A1 WO9918119 A1 WO 9918119A1
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WO
WIPO (PCT)
Prior art keywords
freeze
protein
multimer
organic acid
methionine
Prior art date
Application number
PCT/JP1998/004416
Other languages
English (en)
Japanese (ja)
Inventor
Kensaku Akita
Kenji Mitsushima
Masayoshi Inoue
Original Assignee
Shionogi & Co., Ltd.
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 Shionogi & Co., Ltd. filed Critical Shionogi & Co., Ltd.
Priority to AU92807/98A priority Critical patent/AU9280798A/en
Publication of WO1999018119A1 publication Critical patent/WO1999018119A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof

Definitions

  • the present invention relates to a method for freeze-drying a protein-containing solution, and more particularly to a method for freeze-drying an L-methionine arylase-containing solution.
  • a method for preserving a protein used as a medicine in a stable state for a long time has been long desired, and the freeze-drying method is one of the effective means.
  • a method for subjecting a solution containing a protein to freeze-drying for example, Japanese Patent Application Laid-Open No. 7-227282 is known.
  • L-methionine-lyase-containing solution contains (1) an oligosaccharide, (2) a water-soluble polysaccharide, (3) a sugar alcohol, (4) a protein, (5) an acidic amino acid, (6) One or more members selected from the group consisting of gluyuthione and (7) N-acetyl cysteine coexist, and freeze-drying prevents long-term reduction of L-methionine lyase activity. A method for doing so is disclosed. However, in this patent application, there is no report on suppression of a multimer formed during freeze-drying, and the organic acid metal salt used in the present invention is not included as an additive.
  • Purified L-methionine arylase and L-asparaginase are usually present in the solution in tetrameric form.
  • a solution containing such a protein especially a solution containing L-methionine lyase
  • the tetramer associates to form a multimer of 8 or more. Is formed.
  • An object of the present invention is to suppress the formation of a polymer by associating a stable and biologically active minimum unit protein during lyophilization.
  • the formation of the multimer needs to be suppressed for the following reasons.
  • One possibility is that the higher the molecular weight of the multimer formed, the higher the antigenicity may be. In other words, there is a possibility that the pharmaceutical protein itself may become an allergen, and it is necessary to prevent this.
  • the second reason is that high quality preservation is required for drugs used as pharmaceuticals, and it is desirable that the quality before and after freeze-drying be constant.Before and after freeze-drying, there is a difference in quality due to the formation of multimers. Is not preferred. Therefore, it is necessary to suppress the formation of multimers. However, it has recently been the case that pharmaceutical protein preparations have been commercialized, and there have been few attempts to suppress the formation of these multimers.
  • the present invention provides a method for easily producing a preparation containing a high-purity protein in which formation of a multimer is suppressed even when freeze-dried, and a method for freeze-drying a protein-containing solution.
  • the present inventors have conducted intensive studies to achieve the above object, and as a result, in a method for freeze-drying a protein-containing solution, particularly a L-methionine r-lyase-containing solution, the metal salt of an organic acid was added to the solution before freeze-drying.
  • the present inventors have found that the addition of a salt can suppress the formation of a multimer of octamer or more protein even after lyophilization and subsequent dissolution, thus completing the present invention.
  • the present invention provides a method for freeze-drying a solution containing a stable and biologically active minimum unit of a protein and a metal salt of an organic acid, and suppressing the protein from associating during lyophilization to form a multimer.
  • a method for producing a freeze-dried product containing the protein a method for producing a freeze-dried product of the present invention, wherein the protein is any of L-methionine lyase, L-asparaginase, and interleukin-2;
  • a method for producing the freeze-dried product of the present invention wherein the smallest protein that is stable and biologically active is a tetrameric L-methionine-lyase, and the multimer is an 8-mer or more multimer;
  • organic acid is citric acid, acetic acid, lactic acid or dalconic acid
  • metal salt of the organic acid is sodium citrate, sodium acetate, sodium lactate or sodium gluconate
  • a solid pharmaceutical composition comprising a lyophilized product produced by the method of the present invention
  • a solid pharmaceutical composition comprising a lyophilized product containing a stable and biologically active minimum unit protein that forms a multimer during lyophilization and a metal salt of an organic acid that suppresses the formation of the multimer;
  • a solid pharmaceutical composition of the present invention comprising a freeze-dried product containing L-methionine arylase and a metal salt of an organic acid;
  • a method comprising suppressing freeze-drying of the protein to form a multimer by lyophilizing a solution containing a stable and biologically active minimum unit of a protein and an organic acid metal salt. About.
  • the “protein” according to the present invention includes proteins such as L-methionine lyase, L-asparaginase, and interleukin-2.
  • L —methionine r-lyase is an enzyme that uses pyridoxal phosphate (hereinafter abbreviated as PLP) as a coenzyme [Tanaka, H., et al., Biochemistry 16, 100-106 (1977)]. W09 4/1 1 5 3 5 published May 26, 1994].
  • Proteins may not be stable in monomers depending on their type.
  • tetramers composed of the same subunit are stable and have biological activity.
  • stable and biologically active minimum unit protein means a tetramer in L-methionine lyase and L-asparaginase.
  • interleukin_2 is a monomer and has stability. Therefore, in the case of interleukin-2, “stable and biologically active minimum unit” means a monomer.
  • multimer refers to a multimer associated with the aforementioned “stable and biologically active minimum unit protein”. Therefore, when the “protein” is L-methionine lyase or L-asparaginase, “multimer” means an octamer or more multimer. In the case of interleukin-2, “multimer” means a dimer or higher multimer.
  • the protein concentration before lyophilization is in the range of 1-4 Omg / m 1. Since L-methionine lyase is actually used at about 30 mg / ml, it is preferably in the range of 20 to 40 mgZml.
  • the present invention suppresses the formation of multimers by adding an organic acid metal salt as an additive.
  • Organic acids include cunic, dalconic, lactic and acetic acids.
  • the “metal” includes sodium, potassium, calcium, and the like. Preferably, sodium is used.
  • the concentration of the organic acid metal salt when the organic acid metal salt is added as an additive is preferably 1 to 50 OmM. However, considering the use of excipients, Preferably, it is added at 50 to 200 mM.
  • the solution according to the present invention includes a buffer solution.
  • a buffer solution of sodium phosphate (10 mM Na-phosphate buffer, pH 7.2) is used, but another buffer solution may be a buffer solution of a metal salt of citric acid.
  • the solution containing L-methionine arylase preferably uses a sodium phosphate buffer, and the buffer preferably further contains PLP. Since L-methionine ⁇ -lyase is unstable under weakly acidic conditions, the pH of the solution is preferably in the range of 6.5 to 9.0, but the particularly preferred ⁇ ⁇ is 7.0 to 8.0. It is 0.
  • freeze-drying includes the time from the time when the protein-containing solution is freeze-dried using a freeze dryer to the time when the obtained freeze-dried product is dissolved with a buffer or the like.
  • the protein-containing solution to which the organic acid metal salt has been added can be freeze-dried by an ordinary method.
  • freeze-drying was performed for 20 hours using a freeze dryer.
  • the obtained freeze-dried product can be stored at an appropriate temperature, but a lower storage temperature allows a longer storage period, so that it is usually ⁇ 80 to 30 ° C., preferably ⁇ 40 to 1 ° C. It is desirable to store at 0 ° C.
  • Solid pharmaceutical composition means a composition before dissolution including a freeze-dried product of a protein having a pharmaceutical use and a metal salt of an organic acid.
  • the lyophilized protein obtained by the method of the present invention was dissolved in a buffer containing PLP (lOmM Na-phosphate buffer pH 7.2) in the Examples, but it was dissolved in other appropriate buffer or water ( Water for injection).
  • Preparation means a liquid preparation, particularly an injection.
  • Reference Example 1 Purification of L-meteonin lyase
  • an expression plasmid incorporating the structural gene portion of L-methionine arylase was introduced into Escherichia coli, and A strain for expressing methionine arylase is prepared, and the strain is cultured. After culturing, the wet cells are disrupted, and an aqueous solution of polyethyleneimine is added to adjust the final concentration to 0.1-0.2% (w / v) .Then, the cells are treated at 5-25 ° C for 1-20 minutes. The residue is removed to obtain a crude enzyme solution of L_methionine r-lyase.
  • the obtained crude enzyme solution is salted out with ammonium sulfate and centrifuged, and the obtained precipitate is dissolved in a phosphate buffer to obtain an unpurified L-methionine 7 "-lyase solution.
  • Polyethylene glycol at a final concentration of about 8 to 12% (W / V) is added to the unpurified L-methionine ⁇ -lyase solution, and the mixture is stirred at 4 ° C for about 60 minutes, followed by centrifugation. 2. Production of L-methionine arylase crystals
  • SE-HPLC Size Exclusion HPLC
  • Example 1 Addition of an additive to a solution containing L-methionine arylase (1)
  • the crystals of L-methionine arylase obtained in Reference Example 1 were purified using a 100 MP LP (pyridoxalline) solution. Acid) and adjusted so that the concentration of L-methionine lyase was 30 mg / m 1 (at this point, L-methionine lyase was used).
  • the tetramer of lyase is 98.2%, whereas the multimer of 8 or more is 1.8%).
  • two experimental systems were prepared, pH 7.2 and 8.0 of the buffer.
  • the additives shown in Table 1 were separately prepared and added, and after pre-freezing, freeze-dried in a freeze dryer for 20 hours.
  • the lyophilized product was then dissolved in a buffer (10 mM Na-phosphate buffer, pH 7.2) containing PLP10-iM, and the formation of multimers of L-methionine lyase was measured by the method of Reference Example 2. .
  • the results are shown in Table 1.
  • Example 2 Addition of an additive to a solution containing L-methionine lyase (2) To examine the effect of the concentration of L-methionine lyase on multimer formation, in Example 1, the concentration of lymethionine r -lyase was examined. Is 3 O mg / m 1 On the other hand, in Example 2, the concentration was adjusted to 1 mg / m 1.
  • An object of the present invention is to freeze-dry a solution containing a minimum unit of a stable and biologically active protein and a metal salt of an organic acid, and to suppress the formation of a multimer associated with the protein during freeze-drying.
  • a method for producing the freeze-dried product containing the protein According to the present invention, it is possible to provide a highly pure drug in which the formation of multimers is suppressed even during freeze-drying.
  • the present invention provides a production method useful for formulating a drug containing L-methionine lyase.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé de préparation de produits lyophilisés contenant des protéines, le procédé consistant à lyophiliser une solution contenant une unité minimale d'une protéine stable, biologiquement active et un sel métallique d'un acide organique, tout en empêchant la protéine de s'associer à un élément pour former un polymère pendant l'opération de lyophilisation.
PCT/JP1998/004416 1997-10-03 1998-09-30 Procede de lyophilisation de proteines WO1999018119A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU92807/98A AU9280798A (en) 1997-10-03 1998-09-30 Process for freeze-drying proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP9/270705 1997-10-03
JP27070597 1997-10-03

Publications (1)

Publication Number Publication Date
WO1999018119A1 true WO1999018119A1 (fr) 1999-04-15

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WO (1) WO1999018119A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60120823A (ja) * 1983-12-02 1985-06-28 Green Cross Corp:The ＩｇＧ単量体
JPS62164631A (ja) * 1986-01-07 1987-07-21 塩野義製薬株式会社 インタ−ロイキン−２組成物
JPH01246226A (ja) * 1988-03-28 1989-10-02 Sumitomo Pharmaceut Co Ltd 安定な修飾アスパラギナーゼ含有組成物
JPH03504721A (ja) * 1988-04-13 1991-10-17 シタス　コーポレイション 腫瘍壊死因子配合物
JPH05345728A (ja) * 1991-12-12 1993-12-27 Roussel Uclaf 非グリコシル化還元型組換えヒトｉｌ２の安定化医薬組成物及びその製造方法
JPH07227282A (ja) * 1993-12-24 1995-08-29 Wako Pure Chem Ind Ltd Ｌ−メチオニン γ−リアーゼの安定化法
JPH09182592A (ja) * 1995-10-30 1997-07-15 Shionogi & Co Ltd 組換えＬ−メチオニン γ−リアーゼ

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60120823A (ja) * 1983-12-02 1985-06-28 Green Cross Corp:The ＩｇＧ単量体
JPS62164631A (ja) * 1986-01-07 1987-07-21 塩野義製薬株式会社 インタ−ロイキン−２組成物
JPH01246226A (ja) * 1988-03-28 1989-10-02 Sumitomo Pharmaceut Co Ltd 安定な修飾アスパラギナーゼ含有組成物
JPH03504721A (ja) * 1988-04-13 1991-10-17 シタス　コーポレイション 腫瘍壊死因子配合物
JPH05345728A (ja) * 1991-12-12 1993-12-27 Roussel Uclaf 非グリコシル化還元型組換えヒトｉｌ２の安定化医薬組成物及びその製造方法
JPH07227282A (ja) * 1993-12-24 1995-08-29 Wako Pure Chem Ind Ltd Ｌ−メチオニン γ−リアーゼの安定化法
JPH09182592A (ja) * 1995-10-30 1997-07-15 Shionogi & Co Ltd 組換えＬ−メチオニン γ−リアーゼ

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CONSTANTINO H. R., LANGER R., KLIBANOV A. M.: "AGGREGATION OF A LYOPHILIZED PHARMACEUTICAL PROTEIN, RECOMBINANT HUMAN ALBUMIN: EFFECT OF MOISTURE AND STABILIZATION BY EXCIPIENTS.", BIOTECHNOLOGY. THE INTERNATIONAL MONTHLY FOR INDUSTRIAL BIOLOGY, NATURE PUBLISHING GROUP, US, vol. 13., no. 05., 1 January 1995 (1995-01-01), US, pages 493 - 496., XP002915356, ISSN: 0733-222X, DOI: 10.1038/nbt0595-493 *
HORA M., RANA R. K., SMITH F. W.: "LYOPHILIZED FORMULATIONS OF RECOMBINANT TUMOR NECROSIS FACTOR.", PHARMACEUTICAL RESEARCH, SPRINGER NEW YORK LLC, US, vol. 09., no. 01., 1 January 1992 (1992-01-01), US, pages 33 - 36., XP002915357, ISSN: 0724-8741, DOI: 10.1023/A:1018919508463 *

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Publication number Publication date
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