WO1994021688A1 - Method of decoloring human serum albumin - Google Patents

Method of decoloring human serum albumin Download PDF

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Publication number
WO1994021688A1
WO1994021688A1 PCT/JP1994/000459 JP9400459W WO9421688A1 WO 1994021688 A1 WO1994021688 A1 WO 1994021688A1 JP 9400459 W JP9400459 W JP 9400459W WO 9421688 A1 WO9421688 A1 WO 9421688A1
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Prior art keywords
serum albumin
human serum
hsa
decolorizing
plasma
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PCT/JP1994/000459
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French (fr)
Japanese (ja)
Inventor
Akinori Sumi
Munehiro Noda
Wataru Ohtani
Takao Ohmura
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The Green Cross Corporation
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Publication of WO1994021688A1 publication Critical patent/WO1994021688A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C07K14/625—Extraction from natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for decolorizing human serum albumin derived from plasma. According to the decolorizing method of the present invention, it is possible to remove a certain coloring component (for example, pyrilvin) in the plasma component. Particularly, it is extremely useful for removing coloring components having an absorption wavelength of around 45 O nm, that is, red coloring components.
  • a certain coloring component for example, pyrilvin
  • HSA human serum albumin
  • HSA human serum albumin
  • This protein is made in the liver and is primarily responsible for maintaining normal osmotic pressure in the bloodstream. It also functions as a carrier for various serum molecules.
  • HS A is administered in various clinical settings. For example, shock and burn patients usually require frequent doses of HSA to restore blood volume and thereby improve some of the symptoms associated with trauma. Patients with hypoproteinemia or fetal erythroblastosis may also require treatment with HSA. Therefore, the basic therapeutic value of administering HSA is to treat conditions such as fluid loss from blood vessels, such as in surgery, shock, burns, and hypoproteinemia that causes edema. It is in the point of doing.
  • HSA is produced primarily as a product from a fraction of the collected blood.
  • Various studies have been conducted on the method of isolating and purifying HSA from plasma, and the method has been put to practical use.
  • corn ethanol fractionation, PEG fractionation, and ammonium sulfate fractionation are known.
  • a method combining an anion exchanger treatment with a heating treatment at 60 ° C. for 10 hours Japanese Patent Laid-Open No. 2-191226), an anion exchanger treatment, a cation exchanger treatment and A method of combining heat treatment at 0 ° C for 10 hours (Japanese Patent Application Laid-Open No. 3-117123) has also been developed.
  • an object of the present invention is to provide a plasma-derived HSA by removing the above-mentioned coloring components, which could not be sufficiently removed by the conventional method for purifying plasma-derived HSA, to sufficiently suppress coloring.
  • disclosure of c invention is to provide a method of decolorizing HS a that can
  • the present inventors have conducted intensive studies in view of the above circumstances, and as a result, by treating plasma-derived HSA with a chelating resin, it was possible to remove the coloring components and sufficiently suppress the coloring of HSA. And completed the present invention.
  • the method for decolorizing HS A of the present invention is preferably a method for purifying HS A derived from plasma, particularly preferably at the end of the step. It is characterized by being treated with a resin.
  • the source of albumin which is the starting material for the method of the present invention, is not particularly limited, but those derived from human are particularly used.
  • a starting material for preparing albumin for example, Fraction V obtained by cold alcohol fractionation by Kohn is exemplified.
  • HSA can be obtained through the following steps (1) to (3) according to the sixth method of the cold alcohol fractionation method described by Kohn in JP-A-3-128398.
  • the HSA obtained as described above may be further purified by a known method, such as various fractionation methods, adsorption chromatography, affinity chromatography, gel filtration, density gradient centrifugation, dialysis, etc. It is purified by a known method.
  • the HSA decolorization step is a purification step that can usually be performed on plasma-derived HSA. It is particularly preferably carried out by contacting HS A with a chelating resin incorporated at the end thereof, preferably having a specific exchange group.
  • a chelate resin is a polymer compound in which a chelate ligand (ligand part) capable of forming a complex with a metal ion is introduced into a polymer carrier having a three-dimensional network structure.
  • a weak acid is used.
  • Various chelating resins of the type, polyamine type, aminocarboxylic acid type, thiourea type, aminophosphoric acid type, thiol type or amidoxime type can be used.
  • the chelate resin is synthesized by polymerizing or copolymerizing a monomer capable of forming a chelate, or by introducing a chelate ligand into a polymer chain by a chemical reaction of a functional group of a polymer carrier.
  • the carrier portion of the chelate resin is preferably hydrophobic.
  • a carrier include a copolymer of styrene and divinylbenzene, and a copolymer of acrylic acid and methacrylic acid. And the like.
  • the ligand part is preferably an exchange group containing a nitrogen atom, particularly an exchange group derived from a compound having an amine or Z or imine in the molecule.
  • N-methylglucamine HN (CH 3 ) CH 2 -ECH ( OH) 11 4 CH 2 OH
  • other polyalkylene polyamines such as polyamine (polyethylene polyamine [H 2 N- ⁇ CH 2 CH 2 NH 2 -H]] [H 2 NF (CH 2 NH-CH . 2 ⁇ - m ⁇ H] also included); and Chio urea [H 2 NCSNH 2] force, monovalent groups derived from al least a compound selected, Ru especially include those Amino groups.
  • chelate resins having the above-mentioned carrier portion and ligand portion include DIAION CRB02 in which the carrier portion is a copolymer of styrene and divinylbenzene [ligand portion; _N (CH 3 ) CH 2 -ECH ( OH) ⁇ - 4 CH 2 OH , manufactured by Mitsubishi Kasei], DIAION CR20 [ligand portion; single NH4C H 2 CH 2 NH ⁇ - "H, manufactured by Mitsubishi Kasei], LEWATIT TP214 [ligand portion; _NH CS ⁇ 2, manufactured by Bayer ], Amberlite CG 4000 or the like is preferably used.
  • the conditions for treatment with the above chelate resin are preferably as follows.
  • pH conditions acidic or neutral (pH 3-9, preferably pH 4-7)
  • Time 1 ⁇ 100 hours, preferably 6 ⁇ 24 hours
  • Ion strength 50 mmho or less, preferably 1 to 10 mmho
  • the degree of coloring of HSA is reduced to 1/2 to 1/10.
  • the absorption wavelength near 450 nm that is, the degree of red coloration is reduced to 1/4 to 110.
  • the resulting HSA can be formulated by known methods (eg, heat sterilization at 60 ° C for 10 hours, ultrafiltration, addition of a stabilizer, sterilization filtration, dispensing, lyophilization, etc.). it can.
  • a liquid preparation containing 5 to 25% of HSA, having a pH of about 6.4 to 7.4 and an osmotic pressure ratio of about 1 is exemplified.
  • the HS A preparation thus prepared can be used clinically as an injection, similarly to the plasma-derived HS A preparation.
  • it is used mainly for the purpose of rapidly increasing plasma during shock, replenishing circulating blood volume, improving hypoproteinemia, and maintaining oncotic pressure.
  • it is effective for hypoalbuminemia due to albumin loss (burn, nephrotic syndrome, etc.) and decreased albumin synthesis (cirrhosis, etc.), hemorrhagic shock, etc.
  • It can also be used as a stabilizer, carrier or carrier for pharmaceuticals.
  • HSA treated by the decolorizing method of the present invention is usually administered to adults once a 20% to 5% (5 to 12.5 g as HSA) slowly or intravenously by intravenous drip infusion of HSA 25% solution. It is adjusted according to body weight.
  • the coloring degree before chelating resin treatment (A 45. Nm / A 28 o nm ) was 0.0087, so the coloring degree after chelating resin treatment (A 450 nm / A 280 perennialJ It can be seen that it was reduced to 1-7 before processing.
  • albumin-midori was used for the cryogenic ethanol fractionation of corn using only healthy human plasma screened for HBs antigen and anti- ⁇ 1 IV antibody and screened by ALT (GPT) value.
  • a HSA fraction having a purity of 96% or more obtained by separating and purifying by HPLC was adjusted to an albumin concentration of 25% (w / v). This is a preparation that has been subjected to heat treatment for a long time.
  • the method for decolorizing HSA of the present invention it is possible to remove certain coloring components (for example, bilirubin) in plasma components that could not be sufficiently removed by a conventional method for purifying plasma-derived HSA. .
  • the decolorizing method of the present invention is extremely useful for removing coloring components near the absorption wavelength of 45 O nm, that is, red coloring components.c Therefore, the present invention provides HS A whose coloring is sufficiently suppressed. Can get

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Abstract

A human serum albumin (HSA) derived from the plasma is treated with a chelate resin, preferably one having a nitrogenous exchange group as the ligand, in the step of purifying HSA, particularly preferably in the final stage thereof. This method serves to remove some kind of colored components of the plasma, such as bilirubin, which have been difficult to remove satisfactorily by the conventional method of purifying plasma-derived HSA. It is particularly useful for removing colored components having an absorption wavelength around 450 nm, i.e., red-colored components, thus giving HSA sufficiently reduced in coloration.

Description

明 細 書  Specification
ヒト血清アルブミンの脱色方法  Decolorization method for human serum albumin
技術分野  Technical field
本発明は、 血漿由来のヒト血清アルブミンの脱色方法に関する。 本発明の脱色 方法によれば、 血漿成分中のある種の着色成分 （例えばピリルビン） を除去する ことができる。 特に吸収波長 4 5 O nm付近の着色成分、 すなわち赤色系の着色 成分の除去に極めて有用である。  The present invention relates to a method for decolorizing human serum albumin derived from plasma. According to the decolorizing method of the present invention, it is possible to remove a certain coloring component (for example, pyrilvin) in the plasma component. Particularly, it is extremely useful for removing coloring components having an absorption wavelength of around 45 O nm, that is, red coloring components.
背景技術  Background art
アルブミン、 特にヒト血清アルブミン （以下 「HSA」 ともいう。 ） は血漿の 主要な蛋白構成成分である。 この蛋白は肝臓中で作られ、 主に血流中で正常な浸 透圧を維持する責を負う。 また種々の血清分子のキヤリア一としての機能を持つ ている。 HS Aは種々の臨床上の状況において投与される。 例えば、 ショックや 熱傷患者では血液量を元に戻し、 それにより外傷に関連するいくつかの症状を改 善させるために、 通常は HSAの頻回投与を必要とする。 低蛋白血症や胎児性赤 芽球症に罹っている患者にも HS Aによる治療を必要とすることがある。 したが つて、 HS Aを投与する基本的な治療上の意義は、 外科手術、 ショック、 火傷、 浮腫を起こす低蛋白血症におけるがごとく、 血管からの液体の損失がある様な状 態を治療する点に存する。  Albumin, especially human serum albumin (HSA) is a major protein component of plasma. This protein is made in the liver and is primarily responsible for maintaining normal osmotic pressure in the bloodstream. It also functions as a carrier for various serum molecules. HS A is administered in various clinical settings. For example, shock and burn patients usually require frequent doses of HSA to restore blood volume and thereby improve some of the symptoms associated with trauma. Patients with hypoproteinemia or fetal erythroblastosis may also require treatment with HSA. Therefore, the basic therapeutic value of administering HSA is to treat conditions such as fluid loss from blood vessels, such as in surgery, shock, burns, and hypoproteinemia that causes edema. It is in the point of doing.
現在、 HSAは、 主として採取した血液の分画からの産物として製造されてい る。 HS Aを血漿から単離、 精製する方法としては各種研究がなされ、 実用化さ れている。 例えば、 コーンのエタノール分画法、 PEG分画法、 硫安分画法など が知られている。 また最近では、 陰イオン交換体処理と 60°C， 1 0時間の加熱 処理とを組み合わせる方法 （特開平 2— 1 9 1 226号公報） 、 陰イオン交換体 処理、 陽イオン交換体処理および 6 0°C, 1 0時間の加熱処理を組み合わせる方 法 （特開平 3— 1 7 1 23号公報） なども開発されている。  Currently, HSA is produced primarily as a product from a fraction of the collected blood. Various studies have been conducted on the method of isolating and purifying HSA from plasma, and the method has been put to practical use. For example, corn ethanol fractionation, PEG fractionation, and ammonium sulfate fractionation are known. Recently, a method combining an anion exchanger treatment with a heating treatment at 60 ° C. for 10 hours (Japanese Patent Laid-Open No. 2-191226), an anion exchanger treatment, a cation exchanger treatment and A method of combining heat treatment at 0 ° C for 10 hours (Japanese Patent Application Laid-Open No. 3-117123) has also been developed.
しかしながら、 上記の単離、 精製方法により得られた HS Aは、 血漿成分中の ある種の着色成分が夾雑し、 この着色成分が H S Aと結合することによつて着色 が発生する。 着色成分が結合した HSAは、 臨床上なんら支障がなくとも、 より —層の品質の向上という観点から、 出来る限り着色を抑えることが望ましい。 したがって、 本発明の目的は、 血漿由来の HSAを得るに際し、 従来の血漿由 来 HS Aの精製方法では充分に除去することができなかった上記着色成分を除去 して、 着色を充分に抑えることができる HS Aの脱色方法を提供することにある c 発明の開示 However, HSA obtained by the above-described isolation and purification methods is contaminated with certain coloring components in the plasma components, and coloring occurs when the coloring components bind to HSA. HSA to which the coloring component is bound, even if there is no clinical problem, —From the viewpoint of improving the quality of the layer, it is desirable to suppress coloring as much as possible. Therefore, an object of the present invention is to provide a plasma-derived HSA by removing the above-mentioned coloring components, which could not be sufficiently removed by the conventional method for purifying plasma-derived HSA, to sufficiently suppress coloring. disclosure of c invention is to provide a method of decolorizing HS a that can
本発明者らは、 上記事情に鑑みて鋭意研究を進めた結果、 血漿由来の HSAを キレート樹脂で処理することにより、 上記着色成分を除去し、 HS Aの着色を充 分に抑えることができることを見出し、 本発明を完成するに到った。  The present inventors have conducted intensive studies in view of the above circumstances, and as a result, by treating plasma-derived HSA with a chelating resin, it was possible to remove the coloring components and sufficiently suppress the coloring of HSA. And completed the present invention.
すなわち、 本発明の HS Aの脱色方法は、 血漿由来の HS Aの精製工程におい て、 特に好ましくはその最後に、 キレート樹脂、 好ましくは窒素原子を含有する 交換基をリガンド部として有するキレ一ト樹脂で処理することを特徴とする。  That is, the method for decolorizing HS A of the present invention is preferably a method for purifying HS A derived from plasma, particularly preferably at the end of the step. It is characterized by being treated with a resin.
(1) 出発原料  (1) Starting material
本発明方法の出発原料であるアルブミンの由来には特に制限がないが、 特にヒ ト由来のものが使用される。 アルブミンを調製するための出発原料としては、 例 えば、 コーン氏の冷アルコール分画によって得られる第 V画分などが例示される。 具体的には、 特開平 3— 1 28 3 9 8号公報に記載されたコーン氏の冷アルコ一 ル分画法第 6法に従って、 以下の①〜③の工程を経て HS Aが得られる。  The source of albumin, which is the starting material for the method of the present invention, is not particularly limited, but those derived from human are particularly used. As a starting material for preparing albumin, for example, Fraction V obtained by cold alcohol fractionation by Kohn is exemplified. Specifically, HSA can be obtained through the following steps (1) to (3) according to the sixth method of the cold alcohol fractionation method described by Kohn in JP-A-3-128398.
① HS A含有溶液をエタノール 4 0 %、 pH4.8、 一 5 °Cで処理して生じた 沈殿を回収する （第 V画分） 。  (1) Treat the HS A-containing solution with ethanol at 40%, pH 4.8, and 15 ° C, and collect the precipitate generated (Fraction V).
② 沈殿を適当な溶媒に溶解し、 エタノール 1 0%、 pH4.5、 一 3°Cで処理 して、 上清を回収する。  (2) Dissolve the precipitate in an appropriate solvent, treat with ethanol 10%, pH 4.5, and 13 ° C, and collect the supernatant.
③ 上清をエタノール 4 0%、 pH5.2、 一 5 °Cで処理して生じた沈殿を回収 し、 HSAを得る。  (3) Treat the supernatant with ethanol 40%, pH 5.2, and 15 ° C, collect the resulting precipitate, and obtain HSA.
以上のようにして得られた H S Aは、 公知の方法によりさらに精製されてもよ く、 各種分画法、 吸着クロマトグラフィー、 ァフィ二ティクロマトグラフィー、 ゲル濾過、 密度勾配遠心分離法、 透析などの公知の方法により精製される。  The HSA obtained as described above may be further purified by a known method, such as various fractionation methods, adsorption chromatography, affinity chromatography, gel filtration, density gradient centrifugation, dialysis, etc. It is purified by a known method.
(2) HSAの脱色  (2) Decolorization of HSA
H S Aの脱色工程は、 血漿由来の H S Aに対して通常行われ得る精製工程にお いて、 特に好ましくはその最後に組み込まれ、 好ましくは特定の交換基を有する キレート樹脂と HS Aとを接触させることにより行われる。 The HSA decolorization step is a purification step that can usually be performed on plasma-derived HSA. It is particularly preferably carried out by contacting HS A with a chelating resin incorporated at the end thereof, preferably having a specific exchange group.
キレート樹脂とは、 三次元網目構造の高分子担体部に、 金属イオンと錯体を形 成し得るキレート配位子 （リガンド部） が導入された高分子化合物であり、 本発 明においては、 弱酸型、 ポリアミン型、 アミノカルボン酸型、 チォ尿素型、 アミ ノリン酸型、 チオール型またはアミ ドォキシ厶型の各種キレート樹脂が使用され 得る。 キレート樹脂は、 キレート生成能を有するモノマーを重合あるいは共重合 させるか、 高分子担体部の官能基の化学反応によって、 高分子鎖内にキレート配 位子を導入して合成される。  A chelate resin is a polymer compound in which a chelate ligand (ligand part) capable of forming a complex with a metal ion is introduced into a polymer carrier having a three-dimensional network structure. In the present invention, a weak acid is used. Various chelating resins of the type, polyamine type, aminocarboxylic acid type, thiourea type, aminophosphoric acid type, thiol type or amidoxime type can be used. The chelate resin is synthesized by polymerizing or copolymerizing a monomer capable of forming a chelate, or by introducing a chelate ligand into a polymer chain by a chemical reaction of a functional group of a polymer carrier.
本発明において、 キレート樹脂の担体部は、 疎水性であることが好ましく、 こ のような担体としては、 例えばスチレンとジビニルベンゼンとの共重合体、 ァク リル酸とメタクリル酸との共重合体などが挙げられる。  In the present invention, the carrier portion of the chelate resin is preferably hydrophobic. Examples of such a carrier include a copolymer of styrene and divinylbenzene, and a copolymer of acrylic acid and methacrylic acid. And the like.
またリガンド部は、 好ましくは窒素原子を含有する交換基、 特にァミンおよび Zまたはィミンを分子内に有する化合物由来の交換基が望ましく、 例えば N—メ チルグルカミン 〔HN (CH3 ) CH2-ECH (OH)十一 4 CH2 OH〕 などの ポリオ一ルイミン；ポリアミン （ポリエチレンボリアミン 〔H2 N-^CH2 CH2 NH -„ H〕 などのポリアルキレンポリアミン 〔H2 N-F(CH2 NH- C H2^-m ΝΗ H〕 も含まれる。 ） ；およびチォ尿素 〔H2 NCSNH2 〕 力、 ら選ばれる少なくとも一化合物由来の一価基、 特にこれらのァミノ基が挙げられ る。 Further, the ligand part is preferably an exchange group containing a nitrogen atom, particularly an exchange group derived from a compound having an amine or Z or imine in the molecule. For example, N-methylglucamine (HN (CH 3 ) CH 2 -ECH ( OH) 11 4 CH 2 OH] and other polyalkylene polyamines such as polyamine (polyethylene polyamine [H 2 N- ^ CH 2 CH 2 NH 2 -H]] [H 2 NF (CH 2 NH-CH . 2 ^ - m ΝΗ H] also included); and Chio urea [H 2 NCSNH 2] force, monovalent groups derived from al least a compound selected, Ru especially include those Amino groups.
上記担体部とリガンド部とを有するキレート樹脂の市販品としては、 担体部分 がいずれもスチレンとジビニルベンゼンとの共重合体である DIAION CRB02 〔リガ ンド部； _N (CH3 ) CH2-ECH (OH) ÷-4 CH2 OH, 三菱化成製〕 、 DIAION CR20 〔リガンド部； 一 NH4C H2 CH2 NH^-„ H, 三菱化成製〕 、 LEWATIT TP214 〔リガンド部； _NH C S ΝΗ2 , バイエル製〕 、 アンバライト CG 4000などが好適に使用される。 Commercially available chelate resins having the above-mentioned carrier portion and ligand portion include DIAION CRB02 in which the carrier portion is a copolymer of styrene and divinylbenzene [ligand portion; _N (CH 3 ) CH 2 -ECH ( OH) ÷ - 4 CH 2 OH , manufactured by Mitsubishi Kasei], DIAION CR20 [ligand portion; single NH4C H 2 CH 2 NH ^ - "H, manufactured by Mitsubishi Kasei], LEWATIT TP214 [ligand portion; _NH CS ΝΗ 2, manufactured by Bayer ], Amberlite CG 4000 or the like is preferably used.
上記キレート樹脂による処理条件は、 好適には次の通りである。  The conditions for treatment with the above chelate resin are preferably as follows.
pH条件：酸性または中性 （pH3〜9、 好ましくは pH4〜7) 時間： 1〜 1 0 0時間、 好ましくは 6〜 24時間 pH conditions: acidic or neutral (pH 3-9, preferably pH 4-7) Time: 1 ~ 100 hours, preferably 6 ~ 24 hours
ィォン強度： 5 0 mm h o以下、 好ましくは 1〜 1 0 mm h o  Ion strength: 50 mmho or less, preferably 1 to 10 mmho
混合比： HSA 2 5 0重量部に対してキレ一ト樹脂 1 0 0〜 1 0万重量部、 好ましくは 1 0 0 0〜 1万重量部 （湿重量）  Mixing ratio: 250 to 100 parts by weight of HSA, 100 to 100,000 parts by weight of the chelating resin, preferably 100 to 10,000 parts by weight (wet weight)
上記のキレート樹脂処理により、 HSAの着色度は 1 /2〜1/1 0に低減さ れる。 特に吸収波長 4 5 0 nm付近、 すなわち赤色系の着色度が 1 / 4〜 1 10 に低減される。  By the above chelate resin treatment, the degree of coloring of HSA is reduced to 1/2 to 1/10. In particular, the absorption wavelength near 450 nm, that is, the degree of red coloration is reduced to 1/4 to 110.
(3) 製剤化  (3) Formulation
得られた HSAは、 公知の手法 （6 0°C 1 0時間の加熱滅菌処理、 限外濾過、 安定化剤の添加、 除菌濾過、 分注、 凍結乾燥など） を経て製剤化することができ る。 該製剤の具体例としては、 HSAを 5〜25%含有し、 pHは 6.4〜7.4程 度、 浸透圧比は 1程度の液状製剤が例示される。  The resulting HSA can be formulated by known methods (eg, heat sterilization at 60 ° C for 10 hours, ultrafiltration, addition of a stabilizer, sterilization filtration, dispensing, lyophilization, etc.). it can. As a specific example of the preparation, a liquid preparation containing 5 to 25% of HSA, having a pH of about 6.4 to 7.4 and an osmotic pressure ratio of about 1 is exemplified.
こうして調製された HS A製剤は、 注射剤として血漿由来 HS A製剤と同様に 臨床上用いることができる。 例えば、 主としてショック時の急速な血漿の増量、 循環血液量の補充、 低蛋白血症の改善、 膠質浸透圧の維持などの目的に使用され る。 具体的な効^ '効果としては、 アルブミンの損失 （熱傷、 ネフローゼ症候群 など） およびアルブミン合成低下 （肝硬変など） による低アルブミン血症、 出血 性ショックなどに有効である。 また、 医薬品の安定化剤あるいは担体、 運搬体と しても利用可能である。  The HS A preparation thus prepared can be used clinically as an injection, similarly to the plasma-derived HS A preparation. For example, it is used mainly for the purpose of rapidly increasing plasma during shock, replenishing circulating blood volume, improving hypoproteinemia, and maintaining oncotic pressure. As specific effects, it is effective for hypoalbuminemia due to albumin loss (burn, nephrotic syndrome, etc.) and decreased albumin synthesis (cirrhosis, etc.), hemorrhagic shock, etc. It can also be used as a stabilizer, carrier or carrier for pharmaceuticals.
本発明の脱色方法で処理された HS Aは、 通常成人 1回、 HSA25%溶液で 20〜5 (HSAとして 5〜12.5 g) を緩徐に静脈内注射または点滴静脈 内投与され、 年令、 症状、 体重により適宜増減される。  HSA treated by the decolorizing method of the present invention is usually administered to adults once a 20% to 5% (5 to 12.5 g as HSA) slowly or intravenously by intravenous drip infusion of HSA 25% solution. It is adjusted according to body weight.
本発明をより詳細に説明するために、 以下に実施例を挙げるが、 本発明はこれ によって何ら限定されるものではない。  The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.
実施例 Example
血漿由来の精製 25 %HS A (商品名 「アルブミン—ミ ドリ」 ミ ドリ十字製） 1 に、 D I A I ON CRB 02 (三菱化成製） 1 gを加え、 p H 6.8、 ィォ ン強度 5mmh 0の条件下、 室温で 24時間攪拌した。 樹脂を蒸留水で洗浄後、 回収された HSA 〔濃度 25% (w/v) 〕 について、 波長 280 nmおよび 450 nmにおける吸光度を測定し、 A45。nra /A28o„m を算出したところ、 0.0012で あつた o 1 g of DIAI ON CRB 02 (manufactured by Mitsubishi Kasei) was added to 25% HS A (trade name: “Albumin-Midori” manufactured by Green Cross) derived from plasma, and pH 6.8, ion strength 5 mmh 0 Under the conditions, the mixture was stirred at room temperature for 24 hours. After washing the resin with distilled water, For recovered HSA [concentration 25% (w / v)], the absorbance was measured at a wavelength of 280 nm and 450 nm, A 45. When nra / A 28 o „m was calculated, it was 0.0012 o
一方、 キレート樹脂処理を行う前の着色度 （A45。nm /A28onm ) は 0,0087で あったことから、 キレート樹脂処理後の着色度 （A 450 nm /A 280„J は、 処理 前の 1ノ 7に低減したことが判る。 On the other hand, the coloring degree before chelating resin treatment (A 45. Nm / A 28 o nm ) was 0.0087, so the coloring degree after chelating resin treatment (A 450 nm / A 280 „J It can be seen that it was reduced to 1-7 before processing.
なお、 上記の 「アルブミン―ミ ドリ」 は、 HB s抗原 ·抗^1 I V抗体が陰性で あり、 かつ ALT (GPT) 値でスクリーニングした健常人血漿のみを用いて、 コーンの低温ェタノール分画法により分離精製して得られた純度 96 %以上の H SA画分を、 アルブミン濃度 25% (w/v) に調整した溶液であって、 肝炎ゥ ィルス不活化のために 60°C, 1 0時間の加熱処理を施した製剤である。  The above-mentioned albumin-midori was used for the cryogenic ethanol fractionation of corn using only healthy human plasma screened for HBs antigen and anti- ^ 1 IV antibody and screened by ALT (GPT) value. A HSA fraction having a purity of 96% or more obtained by separating and purifying by HPLC was adjusted to an albumin concentration of 25% (w / v). This is a preparation that has been subjected to heat treatment for a long time.
産業上の利用可能性  Industrial applicability
本発明の H S Aの脱色方法によれば、 従来の血漿由来 H S Aの精製方法では充 分に除去することができなかった血漿成分中のある種の着色成分 （例えばビリル ビン） を除去することができる。 特に、 本発明の脱色方法によって、 吸収波長 4 5 O nm付近の着色成分、 すなわち赤色系の着色成分の除去に極めて有用である c したがって、 本発明によって、 着色が充分に抑えられた HS Aを得ることができ According to the method for decolorizing HSA of the present invention, it is possible to remove certain coloring components (for example, bilirubin) in plasma components that could not be sufficiently removed by a conventional method for purifying plasma-derived HSA. . In particular, the decolorizing method of the present invention is extremely useful for removing coloring components near the absorption wavelength of 45 O nm, that is, red coloring components.c Therefore, the present invention provides HS A whose coloring is sufficiently suppressed. Can get
^？。 ^? .

Claims

請求の範囲 The scope of the claims
1 . 血漿由来のヒト血清アルブミンをキレート樹脂で処理することを特徴とする ヒト血清アルブミンの脱色方法。  1. A method for decolorizing human serum albumin, comprising treating human serum albumin derived from plasma with a chelating resin.
2 . ヒト血清アルブミンがコーン氏の冷アルコール分画により得られた第 V画分 である請求項 1記載のヒト血清アルブミンの脱色方法。  2. The method for decolorizing human serum albumin according to claim 1, wherein the human serum albumin is the fraction V obtained by the cold alcohol fractionation of Kohn.
3 . キレート樹脂のリガンド部が、 窒素原子を含有する交換基である請求項 1記 載のヒト血清アルブミンの脱色方法。  3. The method for decolorizing human serum albumin according to claim 1, wherein the ligand part of the chelate resin is an exchange group containing a nitrogen atom.
4 . キレート樹脂のリガンド部が、 ァミンおよび Zまたはイミンを有する化合物 由来の交換基である請求項 1記載のヒト血清アルブミンの脱色方法。  4. The method for decolorizing human serum albumin according to claim 1, wherein the ligand part of the chelating resin is an exchange group derived from a compound having an amine, a Z or an imine.
5 . キレート樹脂のリガンド部が、 ポリオ一ルイミン、 ポリアミン、 およびチォ 尿素から選ばれる少なくとも一化合物由来の一価基である請求項 1記載のヒト 血清アルブミンの脱色方法。  5. The method for decolorizing human serum albumin according to claim 1, wherein the ligand part of the chelating resin is a monovalent group derived from at least one compound selected from polyiolimin, polyamine and thiourea.
6 . キレート樹脂による処理条件が以下の条件から選ばれる少なくとも一の条件 を満たす請求項 1記載のヒト血清アルブミンの脱色方法。  6. The method for decolorizing human serum albumin according to claim 1, wherein the treatment conditions with the chelate resin satisfy at least one condition selected from the following conditions.
p H条件：酸性または中性  pH conditions: acidic or neutral
時間： 1〜 1 0 0時間  Time: 1 ~ 100 hours
イオン強度： 5 0 mm h o以下  Ionic strength: 50 mmho or less
混合比： ヒト血清アルブミン 2 5 0重量部に対してキレート樹脂 1 0 0〜  Mixing ratio: Chelate resin 100 to 250 parts by weight of human serum albumin
1 0万重量部 （湿重量）  100,000 parts by weight (wet weight)
7 . キレート樹脂による処理条件が以下の条件から選ばれる少なくとも一の条件 を満たす請求項 1記載のヒト血清アルブミンの脱色方法。  7. The method for decolorizing human serum albumin according to claim 1, wherein the treatment conditions with the chelate resin satisfy at least one condition selected from the following conditions.
p H条件： p H 4〜7  pH condition: pH 4-7
時間： 6〜 2 4時間  Time: 6 to 24 hours
イオン強度： 1〜 1 O mm h o  Ionic strength: 1-1 Ommho
混合比： ヒト血清アルブミン 2 5 0重量部に対してキレート樹脂 1 0 0 0 〜 1万重量部 （湿重量）  Mixing ratio: 100 to 10,000 parts by weight of chelating resin to 250 parts by weight of human serum albumin (wet weight)
PCT/JP1994/000459 1993-03-22 1994-03-22 Method of decoloring human serum albumin WO1994021688A1 (en)

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JP5/62202 1993-03-22

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5188621A (en) * 1975-02-03 1976-08-03
JPH02249458A (en) * 1989-03-24 1990-10-05 Tosoh Corp Deodorization and decoloration of plasma
JPH0523195A (en) * 1991-07-22 1993-02-02 Tosoh Corp Purification of serum albumin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5188621A (en) * 1975-02-03 1976-08-03
JPH02249458A (en) * 1989-03-24 1990-10-05 Tosoh Corp Deodorization and decoloration of plasma
JPH0523195A (en) * 1991-07-22 1993-02-02 Tosoh Corp Purification of serum albumin

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