JP5265514B2 - HGF preparation - Google Patents
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- JP5265514B2 JP5265514B2 JP2009500236A JP2009500236A JP5265514B2 JP 5265514 B2 JP5265514 B2 JP 5265514B2 JP 2009500236 A JP2009500236 A JP 2009500236A JP 2009500236 A JP2009500236 A JP 2009500236A JP 5265514 B2 JP5265514 B2 JP 5265514B2
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- 238000002360 preparation method Methods 0.000 title claims description 66
- 229930006000 Sucrose Natural products 0.000 claims description 42
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 42
- 239000005720 sucrose Substances 0.000 claims description 42
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 17
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 16
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 12
- 235000004279 alanine Nutrition 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 238000009472 formulation Methods 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 4
- 229920000136 polysorbate Polymers 0.000 claims description 4
- 229950008882 polysorbate Drugs 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 description 12
- 239000000872 buffer Substances 0.000 description 12
- 235000001014 amino acid Nutrition 0.000 description 11
- 239000003381 stabilizer Substances 0.000 description 10
- 229920000642 polymer Polymers 0.000 description 9
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 9
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 9
- 229920000053 polysorbate 80 Polymers 0.000 description 9
- 229940068968 polysorbate 80 Drugs 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 3
- 229930195725 Mannitol Natural products 0.000 description 3
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- 239000000600 sorbitol Substances 0.000 description 3
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- 238000012360 testing method Methods 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
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- 239000007921 spray Substances 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
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- 101000993347 Gallus gallus Ciliary neurotrophic factor Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 229940124418 agent for nephritis Drugs 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
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- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
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- 210000004556 brain Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
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- 239000013604 expression vector Substances 0.000 description 1
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- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
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- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 102000057308 human HGF Human genes 0.000 description 1
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- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
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- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Description
本発明は、ＨＧＦ（Hepatic Growth Factor、肝細胞増殖因子）を含有する製剤に関する。 The present invention relates to a preparation containing HGF (Hepatic Growth Factor).
ＨＧＦは、中村らにより発見された、成熟肝細胞に対して最も強力な増殖促進活性を持つ生理活性ペプチドであり（例えば、非特許文献１参照）、近年生物工学的手法により量産が可能になった（例えば、非特許文献２参照）。このＨＧＦは、肝炎や肝硬変のみならず、腎炎や癌などに対する治療・予防薬として、また制癌剤の副作用抑制剤や創傷治癒剤などへの適用も期待されている。
ＨＧＦの製剤としては、特許文献１に、ＨＧＦにアルブミン、ヒト血清、ゼラチン、ソルビトール、マンニトール、キシリトールなどを安定化剤として含有させた水溶液製剤が開示されている。しかしながら、前記ＨＧＦ水溶液製剤は保存中にＨＧＦが凝集、白濁、ゲル化が進行するという難点があり、また重合体が形成されるなど物理化学的安定性が低く、生物活性が低下するという問題がある。HGF is a bioactive peptide discovered by Nakamura et al. That has the strongest growth promoting activity on mature hepatocytes (see, for example, Non-Patent Document 1), and in recent years, mass production is possible by biotechnological techniques. (For example, refer nonpatent literature 2). This HGF is expected to be applied not only to hepatitis and cirrhosis, but also as a therapeutic / preventive agent for nephritis, cancer, etc., and as a side effect inhibitor of an anticancer agent, a wound healing agent, and the like.
As a preparation for HGF, Patent Document 1 discloses an aqueous solution preparation containing albumin, human serum, gelatin, sorbitol, mannitol, xylitol and the like as stabilizers in HGF. However, the HGF aqueous solution preparation has the drawbacks that HGF aggregates, becomes cloudy and gels during storage, and has a problem that the physicochemical stability is low, such as formation of a polymer, and biological activity is reduced. is there.
この問題を解決するために、特許文献２には、ＨＧＦにアルギニン、リジン、ヒスチジン、グルタミン、プロリン、グルタミン酸、アスパラギン酸などを安定化剤として含有させた凍結乾燥製剤が開示されている。また、特許文献３には、ＨＧＦにグリシン、アラニン、ソルビトール、マンニトール、デキストラン硫酸などを安定化剤として含有させた凍結乾燥製剤が開示されている。
上記の凍結乾燥製剤は、ＨＧＦの安定化をある程度達成することができるが、さらに安定化効果のよいＨＧＦ製剤が望まれている。
Although the above lyophilized preparation can achieve stabilization of HGF to some extent, an HGF preparation having a better stabilizing effect is desired.
本発明は、従来のＨＧＦ製剤に比べて、長期間の保存でもより安定なＨＧＦ製剤を提供することを目的とする。 An object of the present invention is to provide an HGF preparation that is more stable than a conventional HGF preparation even when stored for a long period of time.
本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、ＨＧＦに精製白糖を添加することにより、ＨＧＦの重合体生成が阻害され、安定なＨＧＦ製剤が得られることを見出し、この知見に基づいてさらに研究を進め、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that the addition of purified sucrose to HGF inhibits HGF polymer production and provides a stable HGF preparation. Based on this knowledge, further research has been conducted and the present invention has been completed.
すなわち、本発明は、
［１］ＨＧＦおよび精製白糖を含有するＨＧＦ製剤、
［２］精製白糖の含有量が、ＨＧＦ１重量部に対して０．０１〜９重量部である前記［１］に記載のＨＧＦ製剤、
［３］さらに、中性アミノ酸を含有する前記［１］または［２］に記載のＨＧＦ製剤、
［４］中性アミノ酸がアラニンである前記［３］に記載のＨＧＦ製剤、
［５］さらに、緩衝剤を含有する前記［１］〜［４］のいずれかに記載のＨＧＦ製剤、
［６］緩衝剤がクエン酸塩である前記［５］に記載のＨＧＦ製剤、
［７］さらに、塩化ナトリウムを含有する前記［１］〜［６］のいずれかに記載のＨＧＦ製剤、
［８］ＨＧＦおよび精製白糖の他に、さらに中性アミノ酸、塩化ナトリウム、緩衝剤および界面活性剤を含有する前記［１］または［２］に記載のＨＧＦ製剤、
［９］中性アミノ酸がアラニンであり、緩衝剤がクエン酸塩であり、界面活性剤がポリソルベートである前記［８］に記載のＨＧＦ製剤、
［１０］凍結乾燥製剤である前記［１］〜［９］のいずれかに記載のＨＧＦ製剤、
［１１］ＨＧＦに精製白糖を添加してＨＧＦ重合体生成を抑制することを特徴とするＨＧＦの安定化方法、および
［１２］精製白糖の添加量がＨＧＦ１重量部に対して０．０１〜９重量部である前記［１１］に記載の安定化方法、
に関する。That is, the present invention
[1] HGF preparation containing HGF and purified sucrose,
[2] The HGF preparation according to [1], wherein the content of purified sucrose is 0.01 to 9 parts by weight with respect to 1 part by weight of HGF,
[3] The HGF preparation according to [1] or [2], further containing a neutral amino acid,
[4] The HGF preparation according to the above [3], wherein the neutral amino acid is alanine,
[5] The HGF preparation according to any one of [1] to [4], further containing a buffer,
[6] The HGF preparation according to the above [5], wherein the buffer is citrate.
[7] The HGF preparation according to any one of [1] to [6], further containing sodium chloride,
[8] The HGF preparation according to [1] or [2] above, which further contains a neutral amino acid, sodium chloride, a buffer and a surfactant in addition to HGF and purified sucrose.
[9] The HGF preparation according to [8], wherein the neutral amino acid is alanine, the buffer is citrate, and the surfactant is polysorbate,
[10] The HGF preparation according to any one of [1] to [9], which is a lyophilized preparation,
[11] A method for stabilizing HGF, wherein purified sucrose is added to HGF to suppress HGF polymer formation, and [12] the amount of purified sucrose added is 0.01 to 9 parts per 1 part by weight of HGF. The stabilization method according to [11] above, which is part by weight,
About.
本発明のＨＧＦ製剤は、長期間保存しても、従来のＨＧＦ製剤に比べてより安定であるという効果を有する。 The HGF preparation of the present invention has an effect that it is more stable than a conventional HGF preparation even when stored for a long period of time.
本発明は、ＨＧＦおよび精製白糖を含有してなるＨＧＦ製剤である。 The present invention is an HGF preparation comprising HGF and purified sucrose.
本発明の有効成分であるＨＧＦは、医薬として使用できる程度に精製されたものであれば、種々の方法で調製されたものを用いることができる。また、本発明に用いるＨＧＦはアミノ酸５残基が欠失したデリーションタイプ（ｄＬｅＨＧＦ）であってもよい。
ＨＧＦの調製方法としては、各種の方法が知られており、例えば、ラット、ウシ、ウマ、ヒツジなどの哺乳動物の肝臓、脾臓、肺臓、骨髄、脳、腎臓、胎盤などの臓器、血小板、白血球などの血液細胞や血漿、血清などから抽出、精製して得ることができる。また、ＨＧＦを産生する初代培養細胞や株化細胞を培養し、培養物（培養上清、培養細胞など）から分離精製してＨＧＦを得ることもできる。あるいは遺伝子工学的手法によりＨＧＦをコードする遺伝子を適切なベクターに組込み、これを適当な宿主に挿入して形質転換し、この形質転換体の培養物から目的とする組換えＨＧＦを得ることができる(例えば、Nature,342,440,1989など参照)。上記の宿主細胞は特に限定されず、従来から遺伝子工学的手法で用いられている各種の宿主細胞、例えば大腸菌、枯草菌、酵母、糸状菌、植物または動物細胞などを用いることができる。As the HGF which is an active ingredient of the present invention, HGF prepared by various methods can be used as long as it has been purified to a degree that can be used as a medicine. The HGF used in the present invention may be a deletion type (dLeHGF) in which 5 amino acid residues are deleted.
Various methods are known as methods for preparing HGF, such as livers, spleens, lungs, bone marrow, brains, kidneys, placenta and other organs of mammals such as rats, cows, horses, sheep, platelets, leukocytes, etc. It can be obtained by extraction and purification from blood cells such as plasma, serum, etc. Alternatively, HGF can be obtained by culturing primary cultured cells or established cells that produce HGF, and separating and purifying them from the culture (culture supernatant, cultured cells, etc.). Alternatively, a gene encoding HGF can be incorporated into an appropriate vector by genetic engineering techniques, inserted into an appropriate host, and transformed, and the desired recombinant HGF can be obtained from the culture of this transformant. (See, for example, Nature, 342, 440, 1989). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells can be used.
より具体的には、ＨＧＦを生体組織から抽出精製する方法としては、例えば、ラットに四塩化炭素を腹腔内投与し、肝炎状態にしたラットの肝臓を摘出して粉砕し、Ｓ−セファロース、ヘパリンセファロースなどのゲルカラムクロマトグラフィー、ＨＰＬＣなどの通常の蛋白質精製法にて精製することができる。また、遺伝子組換法を用い、ヒトＨＧＦのアミノ酸配列をコードする遺伝子を、ウシパピローマウィルスＤＮＡなどのベクターに組み込んだ発現ベクターによって動物細胞、例えば、チャイニーズハムスター卵巣（ＣＨＯ）細胞、マウスＣ１２７細胞、サルＣＯＳ細胞などを形質転換し、その培養上清より得ることができる。 More specifically, as a method for extracting and purifying HGF from a living tissue, for example, carbon tetrachloride is intraperitoneally administered to a rat, and the liver of a rat in a hepatitis state is extracted and pulverized, and S-sepharose, heparin It can be purified by usual protein purification methods such as gel column chromatography such as Sepharose and HPLC. In addition, using genetic recombination methods, animal cells such as Chinese hamster ovary (CHO) cells, mouse C127 cells, and the like by expression vectors in which a gene encoding the amino acid sequence of human HGF is incorporated into a vector such as bovine papillomavirus DNA, Monkey COS cells and the like can be transformed and obtained from the culture supernatant.
本発明の精製白糖は、第十五改正日本薬局方に収載されている精製白糖を安定化剤として好適に使用することができる。精製白糖の添加量は、ＨＧＦ１重量部に対して、０．０１〜９重量部が好ましく、特に０．１〜５重量部の範囲が好ましいが、下限のより好ましい値は０．５重量部であり、上限のより好ましい値は４重量部、さらに好ましい値は３重量部、特に好ましい値は２重量部である。 In the purified sucrose of the present invention, purified sucrose listed in the 15th revision Japanese Pharmacopoeia can be suitably used as a stabilizer. The amount of purified sucrose added is preferably 0.01 to 9 parts by weight with respect to 1 part by weight of HGF, and particularly preferably 0.1 to 5 parts by weight. The more preferred lower limit is 0.5 parts by weight. The upper limit is more preferably 4 parts by weight, still more preferably 3 parts by weight, and particularly preferably 2 parts by weight.
本発明の製剤は種々の製剤形態（例えば、液剤、固形剤、カプセル剤、クリーム剤、スプレー剤など）をとりうるが、一般的には有効成分であるＨＧＦおよび精製白糖のみまたはそれらと慣用の添加物（担体など）と共に水溶性製剤、凍結乾燥製剤などとするのが好ましく、とりわけ凍結乾燥製剤が好ましい。 The preparation of the present invention can take various preparation forms (for example, liquids, solids, capsules, creams, sprays, etc.), but generally only HGF and purified sucrose as active ingredients or those commonly used are used. It is preferable to use water-soluble preparations, freeze-dried preparations and the like together with additives (carriers, etc.), particularly freeze-dried preparations.
本発明のＨＧＦ製剤は、ＨＧＦおよび精製白糖を含有する水溶液とすることで水溶液製剤とすることができ、また該水溶液を通常の凍結乾燥方法で凍結乾燥することでＨＧＦ凍結乾燥製剤を製造できる。前記水溶液における精製白糖の含有量は０．１重量％以上、好ましくは０．５重量％以上であって、９重量％以下、好ましくは５重量％以下、より好ましくは４重量％以下、さらに好ましくは３重量％以下、特に好ましくは２重量％以下である。凍結乾燥製剤における精製白糖の含有量は１０〜８０重量％が好ましく、特に２０〜６０重量％が好ましい。例えば、凍結乾燥製剤は、ＨＧＦを適切な溶剤（例えば、滅菌水、注射用蒸留水、緩衝液、生理食塩水など）に溶解した後、精製白糖を好ましくは０．１〜５重量％、特に好ましくは０．５〜２重量％となるように添加し、必要に応じて、精製白糖以外の安定化剤、緩衝剤、界面活性剤、塩化ナトリウムなどを加え、フィルターなどで濾過して滅菌し、バイアルまたはアンプルに注入して凍結乾燥する。フィルターは、ポアサイズ０．２２μｍ以下の滅菌用フィルターを使用するのが好ましい。滅菌用フィルターとしては、例えば、デュラポア（登録商標、日本ミリポア株式会社製）またはザルトポア２（登録商標、ザルトリウス株式会社製）などが挙げられる。凍結乾燥方法としては、例えば、常圧下で冷却凍結する凍結過程、溶質に拘束されない自由水を減圧下で昇華乾燥する一次乾燥過程、溶質固有の吸着水や結晶水を除去する二次乾燥過程の３つの単位操作による方法が挙げられる。凍結過程の冷却温度は−６０℃〜−４０℃が好ましく、一次乾燥過程の温度は−５０℃〜０℃が好ましく、さらに二次乾燥過程の温度は４℃〜４０℃が好ましい。真空圧力は０．１〜１．５Ｐａが好ましく、特に０．５〜１．２Ｐａが好ましい。凍結乾燥後の乾燥庫内は復圧させる。復圧の方法としては、無菌の空気または不活性ガス（例えば、無菌窒素ガス、無菌ヘリウムガスなど）を庫内に送入して約７０〜１００ｋＰａ、好ましくは約８０〜９５ｋＰａまで一次復圧し、次いで大気圧まで復圧（二次復圧）する方法が好ましい。バイアルの打栓は、一次復圧後に行うのが好ましい。 The HGF preparation of the present invention can be made into an aqueous solution preparation by making it into an aqueous solution containing HGF and purified sucrose, and an HGF lyophilized preparation can be produced by lyophilizing the aqueous solution by an ordinary freeze-drying method. The content of the purified sucrose in the aqueous solution is 0.1% by weight or more, preferably 0.5% by weight or more and 9% by weight or less, preferably 5% by weight or less, more preferably 4% by weight or less, and still more preferably. Is 3% by weight or less, particularly preferably 2% by weight or less. The content of purified sucrose in the lyophilized preparation is preferably 10 to 80% by weight, particularly preferably 20 to 60% by weight. For example, the freeze-dried preparation is prepared by dissolving HGF in a suitable solvent (for example, sterilized water, water for injection, buffer solution, physiological saline, etc.) and then adding purified sucrose, preferably 0.1 to 5% by weight, particularly Preferably, it is added to 0.5 to 2% by weight, and if necessary, a stabilizer other than purified sucrose, a buffer, a surfactant, sodium chloride, etc. are added, and the mixture is filtered and sterilized. Inject into vials or ampoules and lyophilize. The filter is preferably a sterilizing filter having a pore size of 0.22 μm or less. Examples of the filter for sterilization include Durapore (registered trademark, manufactured by Nippon Millipore Corporation) or Zaltopore 2 (registered trademark, manufactured by Sartorius Corporation). The freeze-drying method includes, for example, a freezing process that cools and freezes under normal pressure, a primary drying process that sublimates and drys free water that is not restricted by the solute, and a secondary drying process that removes adsorbed water and crystallization water inherent to the solute. The method by three unit operation is mentioned. The cooling temperature in the freezing process is preferably −60 ° C. to −40 ° C., the temperature in the primary drying process is preferably −50 ° C. to 0 ° C., and the temperature in the secondary drying process is preferably 4 ° C. to 40 ° C. The vacuum pressure is preferably from 0.1 to 1.5 Pa, particularly preferably from 0.5 to 1.2 Pa. Restore the pressure in the drying cabinet after freeze-drying. As a method of restoring pressure, aseptic air or inert gas (for example, aseptic nitrogen gas, aseptic helium gas, etc.) is sent into the chamber, and primary decompression is performed to about 70-100 kPa, preferably about 80-95 kPa, Next, a method of returning to atmospheric pressure (secondary returning pressure) is preferable. The vial is preferably stoppered after the primary decompression.
安定化剤は精製白糖のみでもよいが、精製白糖と共に従来安定化剤として用いられていたグリシン、アラニン、アルギニン、リジン、ヒスチジンなどのアミノ酸、ヘパリン、デキストラン硫酸などの多糖類、ソルビトール、マンニトールなどの糖アルコールなどを好適に使用できる。これらのうち、アミノ酸が好ましく、とりわけアミノ酸のうちグリシン、アラニンなどの中性アミノ酸が好ましい。これらの精製白糖以外の安定化剤の添加量は特に制限されないが、例えばグリシン、アラニンなどの中性アミノ酸を用いる場合の添加量は、精製白糖１重量部に対して、０．０１〜５０重量部が好ましく、０．１〜２０重量部がより好ましい。
安定化剤として、精製白糖と共に中性アミノ酸などの従来の安定化剤を併用することにより、精製白糖のみを用いる場合に比べて安定性をより向上させることができる。The stabilizer may be only purified sucrose, but amino acids such as glycine, alanine, arginine, lysine and histidine, which have been used as stabilizers together with purified sucrose, polysaccharides such as heparin and dextran sulfate, sorbitol, mannitol, etc. Sugar alcohol and the like can be preferably used. Of these, amino acids are preferred, and neutral amino acids such as glycine and alanine are particularly preferred among the amino acids. The addition amount of a stabilizer other than these purified sucrose is not particularly limited. For example, the addition amount in the case of using a neutral amino acid such as glycine or alanine is 0.01 to 50 wt. Part is preferable, and 0.1 to 20 parts by weight is more preferable.
By using a conventional stabilizer such as a neutral amino acid together with purified sucrose as a stabilizer, the stability can be further improved as compared with the case where only purified sucrose is used.
本発明で用いられる緩衝剤としては、例えばリン酸緩衝液、クエン酸緩衝液などが挙げられる。緩衝剤は、再溶解後の水溶液のｐＨを調整しＨＧＦの溶解性を保つ作用を有する。緩衝剤は、再溶解後の水溶液のｐＨが４．５〜６．５となるものが好ましい。緩衝剤として好ましいものは、クエン酸緩衝液が挙げられ、特に好ましくはクエン酸ナトリウム緩衝液が挙げられる。このクエン酸緩衝液は、再溶解後の水溶液中でのＨＧＦの安定化にも寄与する。緩衝剤の添加量は、凍結乾燥製剤を製造する際の凍結乾燥直前の水溶液中の濃度が、１〜１００ｍＭの範囲となるようにするのが好ましい。 Examples of the buffer used in the present invention include phosphate buffer and citrate buffer. The buffer has an action of adjusting the pH of the aqueous solution after redissolving and maintaining the solubility of HGF. The buffer is preferably one having a pH of the aqueous solution after re-dissolution of 4.5 to 6.5. Preferred examples of the buffer include citrate buffer, and particularly preferred is sodium citrate buffer. This citrate buffer also contributes to the stabilization of HGF in the aqueous solution after re-dissolution. The amount of the buffer added is preferably such that the concentration in the aqueous solution immediately before lyophilization when producing a lyophilized preparation is in the range of 1 to 100 mM.
本発明で用いられる界面活性剤としては、例えばポリソルベート２０、ポリソルベート８０、プルロニックＦ−６８、ポリエチレングリコールなどが挙げられ、二種以上を併用してもよい。界面活性剤として特に好ましくは、ポリソルベート系界面活性剤が好ましく、とりわけポリソルベート８０が好ましい。ＨＧＦが容器の材質であるガラスや樹脂などに吸着しやすいため、このような界面活性剤を添加することによって、再溶解後のＨＧＦの容器への吸着を防止することができる。界面活性剤の添加量は、凍結乾燥製剤を製造する際の凍結乾燥直前の水溶液中の濃度が、０．００１〜２．０重量％の範囲であるのが好ましい。 Examples of the surfactant used in the present invention include polysorbate 20, polysorbate 80, pluronic F-68, and polyethylene glycol, and two or more kinds may be used in combination. As the surfactant, a polysorbate surfactant is particularly preferable, and polysorbate 80 is particularly preferable. Since HGF is likely to be adsorbed on glass, resin, or the like, which is the material of the container, the addition of such a surfactant can prevent adsorption of HGF to the container after re-dissolution. As for the addition amount of the surfactant, the concentration in the aqueous solution immediately before lyophilization when producing a lyophilized preparation is preferably in the range of 0.001 to 2.0% by weight.
塩化ナトリウムは、ＨＧＦの溶解性を保つ作用を有する。すなわち、例えば実施例で使用した組換ＨＧＦの場合、塩化ナトリウムの添加により組換ＨＧＦの溶解度が向上し、特に３００ｍＭ以上では著しく溶解性が向上する。塩化ナトリウムの添加量は浸透圧比により制限を受けるが、一般的に用いられる注射剤の浸透圧比を示す量でよい。特に医療用または動物薬用注射剤の浸透圧比として許容される浸透圧比１〜３となる量が好ましい。通常、凍結乾燥製剤を製造する際の凍結乾燥直前の水溶液中の塩化ナトリウム濃度が１５０〜１０００ｍＭとすることが好ましい。 Sodium chloride has an action of maintaining the solubility of HGF. That is, for example, in the case of the recombinant HGF used in the examples, the solubility of the recombinant HGF is improved by adding sodium chloride, and the solubility is remarkably improved particularly at 300 mM or more. The amount of sodium chloride added is limited by the osmotic pressure ratio, but may be an amount that indicates the osmotic pressure ratio of a commonly used injection. In particular, an amount that achieves an osmotic pressure ratio of 1 to 3 that is acceptable as an osmotic pressure ratio of a medical or veterinary injection is preferable. Usually, it is preferable that the sodium chloride concentration in the aqueous solution immediately before freeze-drying when producing a freeze-dried preparation is 150 to 1000 mM.
本発明においては、製剤化に必要な他の添加剤、例えば、溶解補助剤、酸化防止剤、無痛化剤、等張化剤などを含んでもよい。 In the present invention, other additives necessary for formulation, for example, solubilizers, antioxidants, soothing agents, tonicity agents and the like may be included.
上記の如くして得られる本発明の製剤、例えば凍結乾燥製剤は、使用に当たっては、ＨＧＦ濃度が０．１〜４０ｍｇ／ｍＬとなるように注射用蒸留水に溶解し、注射液として用いることができる。さらに、凍結乾燥製剤を含有するクリーム剤、スプレー剤などの外用剤とすることもできる。 The preparation of the present invention obtained as described above, for example, a freeze-dried preparation, can be used as an injection solution by dissolving in distilled water for injection so that the HGF concentration is 0.1 to 40 mg / mL. it can. Furthermore, it can also be used as external preparations such as creams and sprays containing lyophilized preparations.
以下に実施例を用いて本発明を説明するが、本発明はこれらに限定されるものではない。なお、本実施例においては、ＨＧＦとして５アミノ酸欠失型ＨＧＦを用いた。ＨＧＦの重合体の面積百分率（％）（以下、重合体含量（％）という）は高速液体クロマトグラフィー（ＨＰＬＣ）にて定量した値を用いて下記式１により求めた。 The present invention will be described below with reference to examples, but the present invention is not limited to these examples. In this example, 5-amino acid deletion type HGF was used as HGF. The area percentage (%) of the polymer of HGF (hereinafter referred to as polymer content (%)) was determined by the following formula 1 using a value quantified by high performance liquid chromatography (HPLC).
（ＨＰＬＣ条件）
カラム：ゲルろ過カラム（商品名：Ｓｕｐｅｒｄｅｘ ２００ １０／３００、アマシャムバイオサイエンス社製）
移動相：塩化ナトリウム５８．４４ｇ、クエン酸三ナトリウム二水和物２．９４ｇ、ポリソルベート８０ ０．１ｇを水に溶かし、１Ｌとした液をＡ液とする。塩化ナトリウム５８．４４ｇ、クエン酸一水和物２．１０ｇ、ポリソルベート８０ ０．１ｇを水に溶かし、１Ｌとした液をＢ液とする。Ａ液にＢ液を加え、ｐＨ６．０に調整後、０．４５μｍのフィルター（商品名：Ｍｉｌｌｃｕｐ−ＨＶ、孔径：０．４５μｍ、ミリポア社製）でろ過し、使用前に脱気する。室温で保存し、２週間以内に使用する。
カラム温度：２５℃
流量：０．５ｍＬ／分
検液注入量：２５μＬ
分析時間：６０分
検出器：吸光光度計
検出波長：２８０ｎｍ
サンプルクーラー：５分
分子量マーカーは、Ｇｅｌ Ｆｉｌｔｒａｔｉｏｎ Ｓｔａｎｄａｒｄ（カタログ番号：１５１−１９０１、Ｂｉｏ−Ｒａｄ社製）バイアル１本に水５００μＬを加え溶解し、少量試液調整用ろ過フィルター（商品名：Ｕｌｔｒａｆｒｅｅ−ＭＣ、孔径：０．４５μｍ、ミリポア社製）でろ過し、２〜８℃で保存し、３ヶ月以内のものを使用する。(HPLC conditions)
Column: Gel filtration column (trade name: Superdex 200 10/300, manufactured by Amersham Biosciences)
Mobile phase: 58.44 g of sodium chloride, 2.94 g of trisodium citrate dihydrate and 0.1 g of polysorbate 80 are dissolved in water, and the solution made up to 1 L is designated as solution A. A solution obtained by dissolving 58.44 g of sodium chloride, 2.10 g of citric acid monohydrate and 0.1 g of polysorbate 80 in water to make 1 L is designated as solution B. Liquid B is added to liquid A, adjusted to pH 6.0, filtered through a 0.45 μm filter (trade name: Millcup-HV, pore size: 0.45 μm, manufactured by Millipore), and degassed before use. Store at room temperature and use within 2 weeks.
Column temperature: 25 ° C
Flow rate: 0.5 mL / min Test solution injection volume: 25 μL
Analysis time: 60 minutes Detector: Spectrophotometer Detection wavelength: 280 nm
Sample cooler: 5 minute molecular weight marker is Gel Filtration Standard (catalog number: 151-1901, manufactured by Bio-Rad), dissolved in 500 μL of water in one vial, and a filter for adjusting a small amount of reagent solution (trade name: Ultrafree-MC , Pore diameter: 0.45 μm, manufactured by Millipore), stored at 2-8 ° C., and used within 3 months.
また、下記実施例および試験例で用いた希釈用緩衝液は下記のように調製した。
（希釈用緩衝液の調製）
塩化ナトリウム１．１６８８ｇ、クエン酸三ナトリウム二水和物２．９４ｇ、ポリソルベート８０ ０．３ｇを超純水に溶かし、全量１Ｌとした液をＡ液とした。塩化ナトリウム１．１６８８ｇ、クエン酸一水和物２．１０ｇ、ポリソルベート８０ ０．３ｇを超純水（超純水製造装置（商品名：ＭｉｌｌｉＱ Ｇｒａｄｉｅｎｔ、ミリポア社製）を用いて調製、以下同じ）に溶かし、全量１Ｌとした液をＢ液とした。Ａ液にＢ液を加えてｐＨ６．０に調整し、希釈用緩衝液（１）とした。
塩化ナトリウム１７．５３ｇ、クエン酸三ナトリウム二水和物２．９４ｇ、ポリソルベート８０ ０．１ｇを超純水に溶かし、全量１Ｌとした液をＣ液とした。塩化ナトリウム１７．５３ｇ、クエン酸一水和物２．１０ｇ、ポリソルベート８０ ０．１ｇを超純水に溶かし、全量１Ｌとした液をＤ液とした。Ｃ液にＤ液を加えてｐＨ６．０に調整し、希釈用緩衝液（２）とした。Moreover, the dilution buffer used in the following Examples and Test Examples was prepared as follows.
(Preparation of dilution buffer)
A solution obtained by dissolving 1.1688 g of sodium chloride, 2.94 g of trisodium citrate dihydrate and 0.3 g of polysorbate 80 in ultrapure water to make a total volume of 1 L was designated as solution A. 1.1688 g of sodium chloride, 2.10 g of citric acid monohydrate, and 0.3 g of polysorbate 80 were prepared using ultrapure water (ultra pure water production apparatus (trade name: MilliQ Gradient, manufactured by Millipore), the same applies hereinafter). The liquid which was dissolved in the total volume of 1 L was designated as B liquid. B liquid was added to A liquid, and it adjusted to pH 6.0, and was set as the buffer solution for dilution (1).
Liquid C was prepared by dissolving 17.53 g of sodium chloride, 2.94 g of trisodium citrate dihydrate and 0.1 g of polysorbate 80 in ultrapure water to make a total volume of 1 L. A solution obtained by dissolving 17.53 g of sodium chloride, 2.10 g of citric acid monohydrate and 0.1 g of polysorbate 80 in ultrapure water to make a total volume of 1 L was designated as solution D. Solution D was added to solution C to adjust the pH to 6.0 to obtain a dilution buffer (2).
［実施例１］
希釈用緩衝液（１）に、５アミノ酸欠失型ＨＧＦ（以下、単にＨＧＦという）を１０ｍｇ／ｍＬとなるように添加し、精製白糖を０．５重量％濃度となるように添加することによって下記表１の組成の溶液が得られた。
本ＨＧＦ凍結乾燥製剤における精製白糖の含有量は、ＨＧＦ１重量部に対して０．５重量部であり、ＨＧＦ凍結乾燥製剤に対して２６．３重量％である。[Example 1]
By adding 5 amino acid deletion type HGF (hereinafter, simply referred to as HGF) to the dilution buffer (1) so as to be 10 mg / mL, and adding purified sucrose to a concentration of 0.5% by weight. A solution having the composition shown in Table 1 below was obtained.
The content of purified sucrose in the present HGF lyophilized preparation is 0.5 parts by weight with respect to 1 part by weight of HGF and 26.3% by weight with respect to the HGF lyophilized preparation.
［実施例２］
精製白糖の添加濃度を１．０重量％とする以外は、実施例１と同様にして、ＨＧＦ凍結乾燥製剤を得た。
本ＨＧＦ凍結乾燥製剤における精製白糖の含有量は、ＨＧＦ１重量部に対して１重量部であり、ＨＧＦ凍結乾燥製剤に対して４１．７重量％である。[Example 2]
An HGF lyophilized preparation was obtained in the same manner as in Example 1 except that the addition concentration of purified sucrose was 1.0% by weight.
The content of purified sucrose in the HGF lyophilized preparation is 1 part by weight with respect to 1 part by weight of HGF and 41.7% by weight with respect to the HGF lyophilized preparation.
［実施例３］
精製白糖の添加濃度を２．０重量％とする以外は、実施例１と同様にして、ＨＧＦ凍結乾燥製剤を得た。
本ＨＧＦ凍結乾燥製剤における精製白糖の含有量は、ＨＧＦ１重量部に対して２重量部であり、ＨＧＦ凍結乾燥製剤に対して５８．８重量％である。[Example 3]
An HGF lyophilized preparation was obtained in the same manner as in Example 1 except that the concentration of purified sucrose was 2.0% by weight.
The content of purified sucrose in the HGF lyophilized preparation is 2 parts by weight with respect to 1 part by weight of HGF and 58.8% by weight with respect to the HGF lyophilized preparation.
［実施例４］
精製白糖の添加濃度を１．０重量％とし、さらにアラニンを５ｍｇ／ｍＬの濃度で添加する以外は、実施例１と同様にして、ＨＧＦ凍結乾燥製剤を得た。
本ＨＧＦ凍結乾燥製剤における精製白糖の含有量は、ＨＧＦ１重量部に対して１重量部であり、ＨＧＦ凍結乾燥製剤に対して３４．５重量％である。[Example 4]
An HGF lyophilized preparation was obtained in the same manner as in Example 1 except that the concentration of purified sucrose was 1.0% by weight and alanine was further added at a concentration of 5 mg / mL.
The content of purified sucrose in the HGF lyophilized preparation is 1 part by weight with respect to 1 part by weight of HGF and 34.5% by weight with respect to the HGF lyophilized preparation.
［比較例１］
添加剤を精製白糖の代わりに、アラニンを２０ｍｇ／ｍＬの濃度で添加する以外は、実施例１と同様にして、ＨＧＦ凍結乾燥製剤を得た。[Comparative Example 1]
An HGF lyophilized preparation was obtained in the same manner as in Example 1 except that alanine was added at a concentration of 20 mg / mL instead of purified sucrose.
［比較例２］
精製白糖を添加しない以外は、実施例１と同様（以下、基本処方という）にして、ＨＧＦ凍結乾燥製剤を得た。[Comparative Example 2]
An HGF freeze-dried preparation was obtained in the same manner as in Example 1 (hereinafter referred to as basic formulation) except that purified sucrose was not added.
［試験例１］
上記実施例および比較例記載の凍結乾燥製剤を５０℃で保存し、１週間後にサンプリングし、タンパク質濃度５ｍｇ／ｍＬになるように希釈用緩衝液（２）で希釈し、ＨＰＬＣを用いて定量後、上記式１から重合体含量（％）を算出した。その結果を下記表３に示す。
表３から明らかなように、基本処方に精製白糖または精製白糖とアラニンを添加した本発明のＨＧＦ製剤は、基本処方または基本処方にアラニンを添加したＨＧＦ製剤に比べて、顕著に重合体含量が抑制されていた。[Test Example 1]
The freeze-dried preparations described in the above Examples and Comparative Examples are stored at 50 ° C., sampled after one week, diluted with a buffer for dilution (2) so that the protein concentration becomes 5 mg / mL, and quantified using HPLC. From the above formula 1, the polymer content (%) was calculated. The results are shown in Table 3 below.
As is apparent from Table 3, the HGF preparation of the present invention in which purified sucrose or purified sucrose and alanine are added to the basic formulation has a significantly higher polymer content than the basic formulation or the HGF formulation in which alanine is added to the basic formulation. It was suppressed.
［試験例２］
希釈用緩衝液（２０ｍＭ クエン酸緩衝液, １Ｍ塩化ナトリウム,ポリソルベート８０ ０．０１重量％）に、ＨＧＦを１０ｍｇ／ｍＬとなるように添加し、精製白糖を０重量％濃度、１重量％濃度、５重量％濃度、１０重量％または２０重量％濃度となるように添加した試料溶液１〜５を各５０μＬ調製した。各試料溶液は、測定に供するまで、約２４時間凍結した。凍結した各試料溶液を再融解し、ＨＧＦの分子量（約８４ｋＤａ）分布を動的光散乱（Ｄｙｎａｍｉｃ Ｌｉｇｈｔ Ｓｃａｔｔｅｒｉｎｇ， ＤＬＳ）法で測定した。測定装置は、蛋白質溶液専用のＰｒｏｔｅｉｎ−Ｓｏｌｕｔｉｏｎ社製Ｄｙｎａ−Ｐｒｏを用いた。測定温度は４℃に設定した。バックグランドには、ＨＧＦを含まない希釈用緩衝液（各５０μＬ）を用いた。各試料溶液中におけるＨＧＦの分子量の多分散度（Ｐｄ％）を表４に示す。
HGF was added to a dilution buffer (20 mM citrate buffer, 1 M sodium chloride, polysorbate 80 0.01 wt%) to a concentration of 10 mg / mL, and purified sucrose was added at a concentration of 0 wt%, 1 wt%, 50 μL each of sample solutions 1 to 5 added so as to have a concentration of 5 wt%, 10 wt%, or 20 wt% was prepared. Each sample solution was frozen for about 24 hours before being subjected to measurement. Each frozen sample solution was thawed, and the molecular weight (about 84 kDa) distribution of HGF was measured by a dynamic light scattering (DLS) method. As the measuring apparatus, Dyna-Pro manufactured by Protein-Solution Co., Ltd., dedicated to the protein solution was used. The measurement temperature was set to 4 ° C. As a background, a dilution buffer (50 μL each) not containing HGF was used. Table 4 shows the molecular weight polydispersity (Pd%) of HGF in each sample solution.
本発明によれば、医薬として有用な保存安定性の優れたＨＧＦ製剤を提供できる。 ADVANTAGE OF THE INVENTION According to this invention, the HGF formulation excellent in the storage stability useful as a pharmaceutical can be provided.
Claims (7)
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| PCT/JP2008/052979 WO2008102849A1 (en) | 2007-02-22 | 2008-02-21 | Hgf preparation |
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| JPS6289628A (en) * | 1985-09-11 | 1987-04-24 | Green Cross Corp:The | Heat treatment of human fibrinogen pharmaceutical |
| JPH0925241A (en) * | 1995-07-11 | 1997-01-28 | Snow Brand Milk Prod Co Ltd | Lyophilized hgf preparation |
| JP2003095975A (en) * | 2001-06-28 | 2003-04-03 | Meiji Milk Prod Co Ltd | Acetate composition of multiple t-cell epitope polypeptide |
| CN1579544A (en) * | 2003-08-09 | 2005-02-16 | 杨喜鸿 | Sucking tablet for promoting liver cell to growth |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6289628A (en) * | 1985-09-11 | 1987-04-24 | Green Cross Corp:The | Heat treatment of human fibrinogen pharmaceutical |
| JPH0925241A (en) * | 1995-07-11 | 1997-01-28 | Snow Brand Milk Prod Co Ltd | Lyophilized hgf preparation |
| JP2003095975A (en) * | 2001-06-28 | 2003-04-03 | Meiji Milk Prod Co Ltd | Acetate composition of multiple t-cell epitope polypeptide |
| CN1579544A (en) * | 2003-08-09 | 2005-02-16 | 杨喜鸿 | Sucking tablet for promoting liver cell to growth |
Non-Patent Citations (1)
| Title |
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| JPN6008019316; 荒川 力: '凍結操作において添加物はどのようにして蛋白質を安定化するのか' 蛋白質核酸酵素 Ｖｏｌ．３７， Ｎｏ．９, 1992, ｐ．１５１７-１５２３ * |
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