JP2011195611A - Purification method for hyaluronic acid and/or salt thereof - Google Patents
Purification method for hyaluronic acid and/or salt thereof Download PDFInfo
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本発明は、「ヒアルロン酸及び／又はその塩」の含有液から「ヒアルロン酸及び／又はその塩」（以下、総称してヒアルロン酸類という〉を分離精製する方法に関する。ヒアルロン酸は化粧品の保湿剤の他、眼科、整形外科、皮膚科等の医薬品として用いられている。 The present invention relates to a method for separating and purifying “hyaluronic acid and / or a salt thereof” (hereinafter collectively referred to as hyaluronic acid) from a liquid containing “hyaluronic acid and / or a salt thereof.” Hyaluronic acid is a moisturizing agent for cosmetics. In addition, it is used as a medicine for ophthalmology, orthopedics, dermatology, etc.
ヒアルロン酸は動物組織、例えば、工業規模ではニワトリのトサカ等から抽出する方法や、微生物を用いた発酵法により製造されている（ジェービー・ウールコック（Ｊ．Ｂ．Ｗｏｏｌｃｏｃｋ）、ジャーナル・オブ・ジェネラルマイクロバイオロジー，８５，３７２−３７５（１９７６））。 Hyaluronic acid is produced by extraction from animal tissues, such as chicken scorpion on an industrial scale, or fermentation using microorganisms (JB Woolcock, Journal of General Microbiology, 85, 372-375 (1976)).
抽出法や発酵法によって製造されるヒアルロン酸には蛋白質や発熱性物質等が不純物として存在し、これらを分離除去して高純度の製品を得る方法が検討されている。特に発熱性物質の除去は非常に困難であり（特開昭５４−６７０２４号公報）、医薬品としても使用できる高純度な製品を得る方法の開発が待たれている。 In hyaluronic acid produced by the extraction method or fermentation method, proteins, pyrogens and the like are present as impurities, and methods for obtaining high-purity products by separating and removing these have been studied. In particular, removal of pyrogens is very difficult (Japanese Patent Laid-Open No. 54-67024), and development of a method for obtaining a high-purity product that can be used as a pharmaceutical is awaited.
例えば、塩化セチルピリジニウム等の第４級アンモニウム塩との錯体を形成させ不純物を分離した後、フロリジルのようなケイ酸マグネシウムのカラムに不純物を吸着させる方法（公表特許昭６２−５０１４７１号公報）がある。また、アニオン交換樹脂を用いて、発酵液から発熱性物質や蛋白質等を除去するヒアルロン酸の精製法も開示されている（特開昭６３−１２２９３号公報）。しかし、これらの方法では、操作が煩雑で収率が低いなどの課題があった。 For example, there is a method of forming a complex with a quaternary ammonium salt such as cetylpyridinium chloride to separate impurities, and then adsorbing the impurities on a column of magnesium silicate such as florisil (published Japanese Patent Publication No. 62-501471). is there. Also disclosed is a method for purifying hyaluronic acid that uses an anion exchange resin to remove pyrogens, proteins, etc. from the fermentation broth (Japanese Patent Laid-Open No. Sho 63-12293). However, these methods have problems such as complicated operation and low yield.
本発明者らは、ヒアルロン酸類を高純度に精製する方法について検討し、発熱性物質、蛋白質、核酸等を除去するためにヒアルロン酸類含有液をアルミナに接触させる方法（特開平１−３１３５０３号公報）、及びヒアルロン酸類含有液をアルミナに接触させた後、シリカゲルに接触させる方法（特開平２−１０３２０４号公報）について提案した。 The present inventors have studied a method for purifying hyaluronic acid with high purity, and a method of bringing a hyaluronic acid-containing solution into contact with alumina in order to remove pyrogens, proteins, nucleic acids, and the like (Japanese Patent Laid-Open No. 1-31503). ), And a method of bringing the hyaluronic acid-containing liquid into contact with alumina and then contacting with silica gel (JP-A-2-103204).
工業的規模で医薬品として使用できる高純度のヒアルロン酸類を得るには、上記の方法においても、（１）不純物のアルミナ、シリカゲルヘの吸着量が少ないので、アルミナやシリカゲルを多量に消費する、（２）アルミナ、シリカゲルは再生使用することができない、（３）ヒアルロン酸の回収率が低いなどの課題が残っていた。 In order to obtain high-purity hyaluronic acids that can be used as pharmaceuticals on an industrial scale, (1) the amount of impurities adsorbed on alumina and silica gel is small, so that a large amount of alumina and silica gel is consumed. 2) Alumina and silica gel cannot be reused, and (3) the recovery rate of hyaluronic acid is low.
本発明の目的は、高収率で高品質な医薬品グレードのヒアルロン酸類を工業的規模で分離精製する方法を提供することにある。 An object of the present invention is to provide a method for separating and purifying high-quality, high-quality pharmaceutical grade hyaluronic acid on an industrial scale.
本発明者等は、ヒアルロン酸類含有液からの発熱性物質や蛋白質等の不純物を効率よく分離除去し、高純度の医薬品のグレードのヒアルロン酸類を精製する新規方法について鋭意研究を重ねた結果、本発明を完成した。 The inventors of the present invention have conducted extensive research on a new method for efficiently separating and removing pyrogenic substances and impurities such as proteins from hyaluronic acid-containing liquids and purifying high-purity pharmaceutical grade hyaluronic acids. Completed the invention.
すなわち、本発明は、以下に記す方法に関するものである。すなわち、
（１）下記のＡ、Ｂ、Ｃ、Ｄ及びＥの５工程を含むことを特徴とするヒアルロン酸及び／又はその塩の精製法。
Ａ工程： 無機吸着剤、又は無機吸着剤と活性炭にヒアルロン酸及び／又はその塩の含有液を接触させる工程。
Ｂ工程： ヒアルロン酸及び／又はその塩の含有液を酸性側のｐＨ範囲内に調整した後、限外ろ過膜にて透析処理する工程。
Ｃ工程： ヒアルロン酸及び／又はその塩の含有液をセラミックス膜に接触させる工程。
Ｄ工程： ヒアルロン酸及び／又はその塩の含有液を天然物を基材としたクロマトグラフィー用充填剤又は、担体に鎖長が炭素数８以上の置換又は未置換の炭化水素基を結合させた逆相クロマトグラフィー用充填剤に接触させる工程。
Ｅ工程： ヒアルロン酸及び／又はその塩の含有液を精密ろ過膜に接触させる工程。
（２）Ａ、Ｂ、Ｃ、Ｄ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（３）Ａ、Ｂ、Ｄ、Ｃ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（４）Ａ、Ｄ、Ｂ、Ｃ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（５）Ａ、Ｃ、Ｂ、Ｄ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（６）Ａ、Ｃ、Ｄ、Ｂ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（７）Ａ、Ｄ、Ｃ、Ｂ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（８）Ｂ、Ａ、Ｃ、Ｄ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（９）Ｂ、Ａ、Ｄ、Ｃ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（１０）Ｂ、Ｄ、Ａ、Ｃ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（１１）Ｄ、Ａ、Ｂ、Ｃ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（１２）Ｄ、Ａ、Ｃ、Ｂ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（１３）Ｄ、Ｂ、Ａ、Ｃ及びＥの工程順に処理することを特徴とする（１）記載のヒアルロン酸及び／又はその塩の精製法、（１４）Ａ工程の無機吸着剤の組成由来がＳｉＯ２、ＭｇＯ、Ａｌ２Ｏ３、Ａｌ（ＯＨ）３、及びＣａＯ等のある適当な配合比よりなるものであることを特徴とする（１）〜（１３）のいずれかに記載のヒアルロン酸及び／又はその塩の精製法、（１５）Ａ工程の無機吸着剤が（１４）に記載の物であり、活性炭が水蒸気賦活活性炭、ウェットな活性炭、粉末活性炭、粒状活性炭、薬品賦活活性炭、石炭由来活性炭、木材由来活性炭、及びドライな活性炭から選ばれた一種であることを特徴とする（１）〜（１３）のいずれかに記載のヒアルロン酸及び／又はその塩の精製法、（１６）Ｂ工程の限外ろ過膜の材質が、酢酸セルロース、ポリアクリロニトリル、ポリスルフォン、ポリエーテルスルフォン、及びポリビニリデンジフルオライドから選ばれたものであることを特徴とする（１）〜（１３）のいずれかに記載のヒアルロン酸及び／又はその塩の精製法、（１７）Ｃ工程のセラミック膜の材質が、Ａｌ２Ｏ３、ＺｒＯ３、ＳｉＯ２、ＴｉＯ２、Ｓｉ３Ｎ４、ＳｉＣ、ＢＮ、ＭｇＯ、及びＺｎＯ等のある適当な配合比によりなることを特徴とする（１）〜（１３）のいずれかに記載のヒアルロン酸及び／又はその塩の精製法、（１８）Ｄ工程の充填剤がセルロース、ヒスチジン、メチルグルタメート、キチンキトサン等の天然物を基材とするクロマトグラフィー用充填剤又は、炭素数８以上の置換又は未置換の炭化水素基特にオクチル基およびこれらの末端を改変した官能基、オクタデシル基およびこれらの末端を改変した官能基から選ばれた一種の官能基を有する逆相クロマトグラフィー用充填剤であることを特徴とする（１）〜（１３）のいずれかに記載のヒアルロン酸及び／又はその塩の精製法、（１９）Ｅ工程の精密ろ過膜が、ポリスルフォン等の親水性膜、６−ナイロン、６，６−ナイロン、７−ナイロン、１１−ナイロン、１２−ナイロン、６，１１−ナイロン、６，１２−ナイロン等からなるポリアミド膜、ポリビニリデンジフルオライド、ポリテトラフロロエチレン、ポリエチレン、ポリプロピレン等からなる疎水性膜、プラスのゼータ電位を有する膜から選ばれたものであることを特徴とする（１）〜（１３）のいずれかに記載のヒアルロン酸及び／又はその塩の精製法、（２０）下記のＡ、Ｂ、Ｃ、Ｄ及びＥの５工程順に処理することを特徴とするヒアルロン酸及び／又はその塩の精製法。
Ａ工程： （１４）記載の無機吸着剤と水蒸気賦活活性炭、ウェットな活性炭、粉末活性炭、粒状活性炭、薬品賦活活性炭、石炭由来活性炭、木材由来活性炭、ドライな活性炭から選ばれた少なくとも一種の混合物にヒアルロン酸及び／又はその塩の含有液を接触させる工程。
Ｂ工程： Ａ工程で得られた溶液を酢酸セルロース、ポリアクリロニトリル、ポリスルフォン、ポリエーテルスルフォン、ハロゲン化ポリオレフィン、ポリアミド、ポリフッ化ビニリデンから選ばれた材質よりなる限外ろ過膜にて酸性側のｐＨ範囲内に調整した後、透析処理する工程。
Ｃ工程： Ｂ工程で得られた溶液をＡｌ２Ｏ３、ＺｒＯ３、ＳｉＯ２、ＴｉＯ２、Ｓｉ３Ｎ４、ＳｉＣ、ＢＮ、ＭｇＯ、及びＺｎＯ等のある適当な配合比の材質よりなるセラミック膜に接触させる工程。
Ｄ工程： Ｃ工程で得られた溶液をセルロース、ヒスチジン、メチルグルタメート、キチンキトサン等の天然物を基材とするクロマトグラフィー用充填剤又は、炭素数８以上の置換又は未置換の炭化水素基特にオクチル基およびこれらの末端を改変した官能基、オクタデシル基およびこれらの末端を改変した官能基から選ばれた一種の官能基を有する逆相クロマトグラフィー用充填剤に接触させる工程。
Ｅ工程： Ｄ工程で得られた溶液をポリスルフォン等からなる親水性膜、６−ナイロン、６，６−ナイロン、７−ナイロン、１１−ナイロン、１２−ナイロン、６，１１−ナイロン、６，１２−ナイロン等からなるポリアミド膜、ポリビニリデンジフルオライド、ポリテトラエチレン、ポリエチレン、ポリプロピレン等からなる疎水性膜、プラスのゼータ電位を有する膜から選ばれたものに接触させる工程。
（２１） ヒアルロン酸の塩がナトリウム塩、カリウム塩、カルシウム塩、及びリチウム塩から選ばれたものである（２０）記載のヒアルロン酸及び／又はその塩の精製法、（２２）ヒアルロン酸及び／又はその塩の含有液の温度が０〜８０℃である（２１）記載のヒアルロン酸及び／又はその塩の精製法、（２３）ヒアルロン酸及び／又はその塩の含有液の濃度が０．１〜１０ｇ／ｌである（２２）記載のヒアルロン酸及び／又はその塩の精製法、（２４）ヒアルロン酸及び／又はその塩の含有液のｐＨがＡ、Ｃ、Ｄ、Ｅ工程においては３〜１２であり、Ｂ工程においては２．５〜５．０である（２３）記載のヒアルロン酸及び／又はその塩の精製法、（２５）前記Ａ工程における無機吸着剤の量がヒアルロン酸及び／又はその塩の含有液１００重量％に対し０〜２０．０重量％である（２４）記載のヒアルロン酸及び／又はその塩の精製法、（２６）前記Ａ工程における活性炭の量がヒアルロン酸及び／又はその塩の含有液１００重量％に対し、５．０〜２０．０重量％である（２５）記載のヒアルロン酸及び／又はその塩の精製法、（２７）前記Ｂ工程における限外ろ過の方法が全量ろ過方式又はクロスフロー方式である（２６）記載のヒアルロン酸及び／又はその塩の精製法、（２８）前記Ｂ工程における限外ろ過膜のろ材形状が平膜、管状膜、スパイラル膜、ホロファイバー（中空糸）膜、プリーツ膜から選ばれた一種である（２７）記載のヒアルロン酸及び／又はその塩の精製法、（２９）前記Ｂ工程における限外ろ過膜の分画分子量が１〜１０万の範囲である（２８）記載のヒアルロン酸及び／又はその塩の精製法、（３０）前記Ｂ工程における限外ろ過処理時の循環又はろ過速度が５ｍ／ｓ以下である（２９）記載のヒアルロン酸及び／又はその塩の精製法、（３１）前記Ｃ工程におけるろ材構造が対称や非対称構造である（３０）記載のヒアルロン酸及び／又はその塩の精製法、（３２）前記Ｃ工程におけるろ材形状が平面膜、管型、カートリッジ、ホロファイバー等から選ばれた一種である（３１）記載のヒアルロン酸及び／又はその塩の精製法、（３３）前記Ｃ工程におけるろ過精度が０．１〜１０μｍの範囲である（３２）記載のヒアルロン酸及び／又はその塩の精製法、（３４）前記Ｃ工程における通液する速度が有効ろ過表面積１ｍ２当たり２００ｌ／ｈｒ以下である（３３）記載のヒアルロン酸及び／又はその塩の精製法、（３５）前記Ｄ工程におけるヒアルロン酸及び／又はその塩の含有液がその他の塩を含むことを特徴とする（３４）記載のヒアルロン酸及び／又はその塩の精製法、（３６）前記Ｄ工程におけるヒアルロン酸及び／又はその塩の含有液がその他の塩が硫酸アンモニウム、クエン酸ナトリウム、クエン酸カリウム、リン酸カリウム、リン酸ナトリウム、硫酸ナトリウム、硫酸マグネシウム、塩化ナトリウム、塩化カリウム、から選ばれた少なくとも一種以上からなる（３５）記載のヒアルロン酸及び／又はその塩の精製法、（３７）前記Ｄ工程におけるヒアルロン酸及び／又はその塩の含有液の塩濃度が０．５〜２０ｗ／ｖ％である（３６）記載のヒアルロン酸及び／又はその塩の精製法、（３８）前記Ｄ工程におけるヒアルロン酸及び／又はその塩の含有液が水系である（３７）記載のヒアルロン酸及び／又はその塩の精製法、（３９）前記Ｄ工程における天然物を基材とするクロマトグラフィー用充填剤又は、逆相クロマトグラフィー用充填剤の担体の形状が球状又は破砕状である（３８）記載のヒアルロン酸及び／又はその塩の精製法、（４０）前記Ｄ工程における天然物を基材とするクロマトグラフィー用充填剤、逆相クロマトグラフィー用充填剤の担体の平均粒径が数μｍ〜約１００μｍである（３９）記載のヒアルロン酸及び／又はその塩の精製法、（４１）前記Ｄ工程における天然物を基材とするクロマトグラフィー用充填剤又は、逆相クロマトグラフィー用充填剤に接触させる方法が、ヒアルロン酸及び／又はその塩の合有液に逆相クロマトグラフィー用充填剤を添加してバッチ式で撹拌するか又は、充填剤を充填塔等に充填後、ヒアルロン酸及び／又はその塩の含有液を通液処理する（４０）記載のヒアルロン酸及び／又はその塩の精製法、（４２）前記Ｅ工程におけるろ材構造が表面ろ過構造又は深層ろ過構造である（４１）記載のヒアルロン酸及び／又はその塩の精製法、（４３）前記Ｅ工程におけるろ材形状がシート、カートリッジ、ホロファイバーから選ばれた一種である（４２）記載のヒアルロン酸及び／又はその塩の精製法、（４４）前記Ｅ工程におけるろ過精度が０．１〜１０μｍの範囲である（４３）記載のヒアルロン酸及び／又はその塩の精製法、（４５）前記Ｅ工程における通液する速度が有効ろ過表面積１ｍ２当たり５００ｌ／ｈｒ以下である（４４）記載のヒアルロン酸及び／又はその塩の精製法、（４６）ヒアルロン酸及び／又はその塩が、ストレプトコッカス・エクイＦＭ−１００を用いて、発酵法により製造されるものであることを特徴とする（１）〜（４５）のいずれかに記載の精製法、（４７）ヒアルロン酸及び／又はその塩が、ストレプトコッカス・エクイＦＭ−３００を用いて、発酵法により製造されるものであることを特徴とする（１）〜（４５）のいずれかに記載の精製法である。
That is, the present invention relates to the method described below. That is,
(1) A method for purifying hyaluronic acid and / or a salt thereof, comprising the following 5 steps of A, B, C, D and E.
Step A: A step of bringing a solution containing hyaluronic acid and / or a salt thereof into contact with the inorganic adsorbent or the inorganic adsorbent and activated carbon.
Step B: A step in which hyaluronic acid and / or its salt-containing liquid is adjusted within the pH range on the acidic side and then dialyzed with an ultrafiltration membrane.
Step C: A step of bringing a liquid containing hyaluronic acid and / or a salt thereof into contact with the ceramic film.
Step D: A substituted or unsubstituted hydrocarbon group having a chain length of 8 or more carbon atoms is bound to a chromatographic packing material or carrier based on hyaluronic acid and / or a salt thereof, or a carrier. Contacting with a packing for reverse phase chromatography.
Step E: A step of bringing a hyaluronic acid and / or salt-containing liquid into contact with a microfiltration membrane.
(2) The method for purifying hyaluronic acid and / or a salt thereof according to (1), wherein the treatment is performed in the order of steps A, B, C, D and E, (3) A, B, D, C and E (1) The method for purifying hyaluronic acid and / or a salt thereof according to (1), wherein the treatment is performed in the order of steps of A, D, B, C, and E (1) (2) Purification method of hyaluronic acid and / or salt thereof according to (1), (5) Purification of hyaluronic acid and / or salt thereof according to (1), which is processed in the order of steps A, C, B, D and E (6) A method of purifying hyaluronic acid and / or a salt thereof according to (1), characterized in that the treatment is performed in the order of steps of (6) A, C, D, B and E, (7) A, D, C, B And (8) a method for purifying hyaluronic acid and / or a salt thereof according to (1) The method for purifying hyaluronic acid and / or a salt thereof according to (1), wherein the treatment is performed in the order of steps of B, A, D, C, and E. (1) The method for purifying hyaluronic acid and / or a salt thereof according to (1), (10) The hyaluronic acid according to (1), wherein the hyaluronic acid is treated in the order of steps B, D, A, C and E (11) purification method of hyaluronic acid and / or salt thereof according to (1), characterized in that the treatment is performed in the order of steps of D, A, B, C and E, (12) (1) The method for purifying hyaluronic acid and / or a salt thereof according to (1), wherein the treatment is performed in the order of steps of D, A, C, B, and E. (13) Steps of D, B, A, C, and E (1) The method for purifying hyaluronic acid and / or a salt thereof according to (1), ) Derived from the composition of the inorganic adsorbent A process SiO 2, MgO, Al 2 O 3, Al (OH) 3, and characterized in that made from an appropriate blending ratio with CaO, etc. (1) - (13) The method for purifying hyaluronic acid and / or a salt thereof according to any one of (13), (15) the inorganic adsorbent in step A is the one described in (14), and the activated carbon is steam activated activated carbon, wet activated carbon, The hyaluronic acid according to any one of (1) to (13), which is a kind selected from powdered activated carbon, granular activated carbon, chemical activated carbon, coal-derived activated carbon, wood-derived activated carbon, and dry activated carbon (16) Whether the material of the ultrafiltration membrane in step B is cellulose acetate, polyacrylonitrile, polysulfone, polyethersulfone, and polyvinylidene difluoride And characterized in that selected (1) to the hyaluronic acid and / or purification of a salt thereof according to any one of (13), the material of the ceramic membrane (17) C step, Al 2 O 3 , ZrO 3 , SiO 2 , TiO 2 , Si 3 N 4 , SiC, BN, MgO, ZnO, etc. (18) The chromatographic packing material based on natural products such as cellulose, histidine, methyl glutamate, chitin chitosan or the like. A substituted or unsubstituted hydrocarbon group, particularly an octyl group, a functional group modified at these ends, an octadecyl group, and a reverse having one kind of functional group selected from functional groups modified at these ends (1) The method for purifying hyaluronic acid and / or a salt thereof according to any one of (1) to (13), wherein the microfiltration membrane in step E is polysulfone Hydrophilic membranes such as 6-nylon, 6,6-nylon, 7-nylon, 11-nylon, 12-nylon, 6,11-nylon, 6,12-nylon, etc., polyvinylidene difluoride The hyaluronic acid according to any one of (1) to (13), wherein the hyaluronic acid is selected from a hydrophobic film made of polytetrafluoroethylene, polyethylene, polypropylene, or the like, or a film having a positive zeta potential And / or a method for purifying the salt thereof, (20) purification of hyaluronic acid and / or a salt thereof, characterized by treatment in the following five-step sequence of A, B, C, D and E .
Step A: At least one mixture selected from the inorganic adsorbent described in (14) and water vapor activated activated carbon, wet activated carbon, powdered activated carbon, granular activated carbon, chemical activated activated carbon, coal-derived activated carbon, wood-derived activated carbon, and dry activated carbon. A step of contacting a liquid containing hyaluronic acid and / or a salt thereof.
Step B: The solution obtained in Step A is subjected to acidic pH in an ultrafiltration membrane made of a material selected from cellulose acetate, polyacrylonitrile, polysulfone, polyether sulfone, halogenated polyolefin, polyamide, and polyvinylidene fluoride. A process of dialysis after adjustment within the range.
Step C: A ceramic made of a material having an appropriate mixing ratio such as Al 2 O 3 , ZrO 3 , SiO 2 , TiO 2 , Si 3 N 4 , SiC, BN, MgO, and ZnO. Contacting the membrane.
Step D: The solution obtained in Step C is a chromatographic filler based on natural products such as cellulose, histidine, methyl glutamate, and chitin chitosan, or a substituted or unsubstituted hydrocarbon group having 8 or more carbon atoms, particularly Contacting with a packing material for reverse phase chromatography having one kind of functional group selected from an octyl group and functional groups modified at these ends, octadecyl group and functional groups modified at these ends.
Step E: The solution obtained in Step D is a hydrophilic film made of polysulfone or the like, 6-nylon, 6,6-nylon, 7-nylon, 11-nylon, 12-nylon, 6,11-nylon, 6, A step of contacting a polyamide film made of 12-nylon or the like, a hydrophobic film made of polyvinylidene difluoride, polytetraethylene, polyethylene, polypropylene, or the like, or a film having a positive zeta potential.
(21) The method for purifying hyaluronic acid and / or a salt thereof according to (20), wherein the salt of hyaluronic acid is selected from sodium salt, potassium salt, calcium salt, and lithium salt, (22) hyaluronic acid and / or Or the method for purifying hyaluronic acid and / or a salt thereof according to (21), wherein the temperature of the solution containing the salt thereof is 0 to 80 ° C., and (23) the concentration of the liquid containing the hyaluronic acid and / or salt thereof is 0.1 (22) The method for purifying hyaluronic acid and / or salt thereof according to (22), wherein the pH of the solution containing hyaluronic acid and / or salt thereof is 3 to 10 g / l in steps A, C, D, and E. (25) The purification method of hyaluronic acid and / or a salt thereof according to (23), which is 2.5 to 5.0 in Step B, (25) The amount of the inorganic adsorbent in Step A is hyaluronic acid and / or Or a salt-containing liquid 1 The purification method of hyaluronic acid and / or a salt thereof according to (24), which is 0 to 20.0% by weight with respect to 00% by weight, (26) the amount of activated carbon in step A includes hyaluronic acid and / or a salt thereof The purification method of hyaluronic acid and / or a salt thereof according to (25), which is 5.0 to 20.0% by weight with respect to 100% by weight of the liquid, (27) The ultrafiltration method in Step B is a total filtration method Or the purification method of hyaluronic acid and / or a salt thereof according to (26), which is a cross flow method, (28) the shape of the filter medium of the ultrafiltration membrane in the step B is a flat membrane, a tubular membrane, a spiral membrane, a hollow fiber (hollow Thread) A method for purifying hyaluronic acid and / or a salt thereof according to (27), which is a kind selected from a membrane and a pleated membrane, and (29) a fractional molecular weight of the ultrafiltration membrane in the step B is 1 to 100,000 Range (28) (30) The hyaluronic acid and / or salt thereof according to (29), wherein the circulation or filtration rate during the ultrafiltration treatment in Step B is 5 m / s or less. (31) The purification method of hyaluronic acid and / or a salt thereof according to (30), wherein the filter medium structure in Step C is a symmetric or asymmetric structure, and (32) the shape of the filter medium in Step C is a flat membrane, tubular type (31) The method for purifying hyaluronic acid and / or a salt thereof according to (31), which is a kind selected from cartridges, holofibers, etc., (33) The filtration accuracy in the step C is in the range of 0.1 to 10 μm (32 ) purification of hyaluronic acid and / or a salt thereof according, (34) the rate of liquid passing in the step C is less effective filtration surface area of 1 m 2 per 200 l / hr (33) according hyaluronic (35) The hyaluronic acid and / or salt thereof according to (34), wherein the solution containing hyaluronic acid and / or salt thereof in step D contains another salt. (36) The liquid containing hyaluronic acid and / or salt thereof in step D is ammonium sulfate, sodium citrate, potassium citrate, potassium phosphate, sodium phosphate, sodium sulfate, magnesium sulfate, (35) The method for purifying hyaluronic acid and / or salt thereof according to (35), comprising at least one selected from sodium chloride and potassium chloride, (37) Salt of hyaluronic acid and / or salt-containing liquid in step D (38) The method for purifying hyaluronic acid and / or a salt thereof according to (36), wherein the concentration is 0.5 to 20 w / v%, (38) The method for purifying hyaluronic acid and / or salt thereof according to (37), wherein the liquid containing hyaluronic acid and / or salt thereof is aqueous, and (39) a packing material for chromatography based on a natural product in the step D Alternatively, the method for purifying hyaluronic acid and / or a salt thereof according to (38), wherein the shape of the carrier for the packing for reverse phase chromatography is spherical or crushed, and (40) the natural product in step D is used as a base material (41) The method for purifying hyaluronic acid and / or a salt thereof according to (39), wherein the average particle size of the carrier for chromatography and the carrier for reverse phase chromatography is several μm to about 100 μm, (41) A method of contacting a natural product-based chromatographic packing material or a reverse phase chromatography packing material with a mixed solution of hyaluronic acid and / or a salt thereof is a reverse phase chromatography. Add a filler for matography and stir in a batch system, or after filling the filler into a packed tower or the like, pass the liquid containing hyaluronic acid and / or its salt through the liquid treatment (40) and (42) The purification method of hyaluronic acid and / or a salt thereof according to (41), wherein the filter medium structure in the E step is a surface filtration structure or a deep layer filtration structure, (43) in the E step The method for purifying hyaluronic acid and / or its salt according to (42), wherein the shape of the filter medium is a kind selected from sheets, cartridges and holofibers, (44) The filtration accuracy in the step E is in the range of 0.1 to 10 μm. some (43) of hyaluronic acid according and / or purification of the salt is (45) the E rate of liquid permeation is less effective filtration surface area of 1 m 2 per 500 l / hr in the step (44 (46) The method for purifying hyaluronic acid and / or a salt thereof described in (46), wherein the hyaluronic acid and / or a salt thereof is produced by fermentation using Streptococcus equii FM-100 ( The purification method according to any one of 1) to (45), (47) hyaluronic acid and / or a salt thereof is produced by fermentation using Streptococcus equi EFM-300. The purification method according to any one of (1) to (45).
本発明の方法によれば、発熱性物貿、タンパク質、微生物、微粒子などの除去された高純度の医薬品グレードのヒアルロン酸類を高収率かつ工業的規模で製造することができる。 According to the method of the present invention, high-purity pharmaceutical grade hyaluronic acids from which pyrogens, proteins, microorganisms, fine particles and the like are removed can be produced in a high yield and on an industrial scale.
以下、更に本発明について詳しく説明する。
本発明に用いられるヒアルロン酸類は、ヒアルロン酸、ヒアルロン酸の塩、又はヒアルロン酸とヒアルロン酸の塩との混合物を包含している。ヒアルロン酸類は、遊離の形でもよく又その塩でもよく例えば、ナトリウム塩、カリウム塩、カルシウム塩、リチウム塩等が挙げられるが、ナトリウム塩が好ましい。さらに本発明で使用するヒアルロン酸類含有液は動物組織から抽出したものでも、また発酵法で製造したものでも使用できる。
The present invention will be further described in detail below.
The hyaluronic acid used in the present invention includes hyaluronic acid, a salt of hyaluronic acid, or a mixture of hyaluronic acid and a salt of hyaluronic acid. Hyaluronic acids may be in a free form or a salt thereof, for example, sodium salt, potassium salt, calcium salt, lithium salt and the like, and sodium salt is preferable. Furthermore, the hyaluronic acid-containing liquid used in the present invention may be extracted from animal tissue or manufactured by fermentation.
発酵法によるヒアルロン酸は例えばストレプトコッカス属のバクテリアを使用して既知の方法で得ることができる。 Hyaluronic acid obtained by fermentation can be obtained by a known method using, for example, bacteria of the genus Streptococcus.
発酵法で使用する菌株は自然界から分離されるストレプトコッカス属等のヒアルロン酸生産能を有する微生物、または特開昭６３−１２３３９２号公報に記載したストレプトコッカス・エクイＦＭ−１００（微工研菌寄第９０２７号）、特開平２−２３４６８９号公報に記載したストレプトコッカス・エクイＦＭ−３００（微工研条寄第２３１９号）のような高収率で安定にヒアルロン酸を生産する変異株が望ましい。発酵液を用いる場合には、既知の方法、例えば、遠心分離やろ過処理等で除菌した液を使用することが望ましい。場合によっては、精密ろ過処理による水不溶微粒子の除去等の操作を行ってもよく、アルコール等の水溶性有機溶媒を添加してヒアルロン酸を析出したものを使用してもよい。また、膜の負荷を軽減する目的でアルミナや活性炭で処理してもよい。 The strain used in the fermentation method is a microorganism having the ability to produce hyaluronic acid such as Streptococcus isolated from nature, or Streptococcus equii FM-100 described in JP-A No. 63-123392 (90 KOJI Bacteria 9027). No.), and Streptococcus equii FM-300 described in Japanese Patent Application Laid-Open No. Hei 2-23489 are desirable, such as a mutant strain that stably produces hyaluronic acid at a high yield. When using a fermentation broth, it is desirable to use a liquid sterilized by a known method such as centrifugation or filtration. Depending on the case, operations such as removal of water-insoluble fine particles by microfiltration may be performed, or a solution obtained by precipitating hyaluronic acid by adding a water-soluble organic solvent such as alcohol may be used. Moreover, you may process with an alumina or activated carbon in order to reduce the load of a film | membrane.
Ａ工程：ヒアルロン酸含有液を無機吸着剤又は無機吸着剤と活性炭等と接触させることにより、タンパク質や発熱性物質等の大量の不純物を効率よく除去する工程。Ａ工程は、プロセスの比較的初期段階にて有効である。 Step A: A step of efficiently removing a large amount of impurities such as proteins and pyrogens by bringing the hyaluronic acid-containing liquid into contact with an inorganic adsorbent or an inorganic adsorbent and activated carbon. Step A is effective in a relatively early stage of the process.
本工程に用いる活性炭は、粉末活性炭、粒状活性炭等の形状に制限されるものではないが、好ましくは、粉末活性炭がよい。又、活性炭の由来（石炭または、ヤシガラ等）、製法（薬品賦活、水蒸気賦活）などに制限されるものではない。又、ドライ炭、ウエット炭等も制限されない。 The activated carbon used in this step is not limited to shapes such as powdered activated carbon and granular activated carbon, but powdered activated carbon is preferable. Moreover, it is not restrict | limited to the origin (coal or coconut husk etc.) of activated carbon, a manufacturing method (chemical activation, steam activation), etc. Also, dry charcoal, wet charcoal and the like are not limited.
活性炭として、ツルミコールＧＬ―３０Ｓ、同ＨＣ―３０Ｓ［粒状、（株）ツルミコール］、白鷺ＷＨ５Ｃ８／３２、同ＬＨ２Ｃ２０／４８、同ＷＨ２Ｃ８／３２ＳＳ［粒状・武田薬品工業（株）］、白鷺Ａ、白鷺Ｐ、精製白鷺、特性白鷺、カルボラフィン［粉状・武田薬品工業（株）］、北越ＳＤ、北越ＧＳＡ［粉状・北越炭素工業（株）］、シルバーＡ、花Ｆ、雪Ａ［粉状・大三工業（株）］等が使用できるが、これらに制限されるものではない。 As activated carbon, Tsurumi Coal GL-30S, HC-30S [granular, Tsurumi Co., Ltd.], Shirasagi WH5C8 / 32, LH2C20 / 48, WH2C8 / 32SS [Granular / Takeda Pharmaceutical Co., Ltd.], Shirasagi A, Shirasagi P, refined white birch, characteristic white birch, carborafin [powder / Takeda Pharmaceutical Co., Ltd.], Hokuetsu SD, Hokuetsu GSA [powder / Hokuetsu Carbon Co., Ltd.], silver A, flower F, snow A [powder -Daisan Kogyo Co., Ltd.] can be used, but is not limited thereto.
本工程に用いる無機吸着剤の組成由来は、Ａｌ２Ｏ３、ＳｉＯ２、ＭｇＯ、ＣａＯ、及びＡｌ（ＯＨ）３等があるがこれらの配合比や、製法、水和物の有無等に制限されるものではない。 The composition of the inorganic adsorbent used in this step is derived from Al 2 O 3 , SiO 2 , MgO, CaO, Al (OH) 3, etc. Is not to be done.
無機吸着剤として、モンモロリナイトの酸処理物から成る活性白土［日本活性白土（株）］、ニッカナイトＧＨＣ―３６、ニッカナイトＧ―３６［日本活性白土（株）］、ニッカゲルＭ―３０、ＳｉＯ２・Ａｌ２Ｏ３から成るニッカゲルＳ−６５［日本活性白土（株）］、Ａｌ２Ｏ３・１０ＳｉＯ２・ｘＨ２Ｏから成るトミックスＡＤ−７００ＮＳ、Ａｌ２Ｏ３・ｘＨ２Ｏから成るトミックスＡＤ−２００ＮＳ、Ａｌ（ＯＨ）３・ＮａＨＣＯ３から成るトミックスＡＤ−４００ＮＳ、６ＭｇＯ・Ａｌ２Ｏ３・ＣＯ２・Ｈ２Ｏから成るトミックスＡＤ―５００ＮＳ、ＭｇＯ・３ＳｉＯ２・ｘＨ２Ｏから成るトミックスＡＤ―６００ＮＳ、ＭｇＯから成るトミックスＡＤ―１００Ｇ、ＭｇＯ・Ａｌ２Ｏ３・２ＳｉＯ２から成るトミックスＡＤ―３００ＮＳＳ、トミックスＡＤ―８００顆粒［富田製薬（株）］、Ｑ―Ｆｉｎｅ２０００、同３０００［富田製薬（株）］、合成ゼオライトＡ―４、合成ゼオライトＦ―９［東洋曹達工業（株）］、天然ゼオライト［日本活性白土（株）］、ベントナイト［和光純薬工業（株）］等が使用できるが、これらに制限されるものではない。 As an inorganic adsorbent, an activated clay made of an acid-treated product of montmorillonite [Nippon Active Soil Co., Ltd.], Nikkanite GHC-36, Nikkanite G-36 [Nippon Active Soil Co., Ltd.], Nikka Gel M-30, Nikkagel S-65 composed of SiO 2 · Al 2 O 3 [Nippon Active Soil Co., Ltd.], Tomix AD-700NS composed of Al 2 O 3 · 10SiO 2 · xH 2 O, and Al 2 O 3 · xH 2 O Tomix AD-200NS, Tomix AD-400NS consisting of Al (OH) 3 · NaHCO 3 , Tomix AD-500NS consisting of 6MgO · Al 2 O 3 · CO 2 · H 2 O, MgO · 3SiO 2 · xH 2 O Tomix AD-600NS, Tomix AD-100G made of MgO, consists MgO · Al 2 O 3 · 2SiO 2 Tomix AD-300NSS, Tomix AD-800 granules [Tonda Pharmaceutical Co., Ltd.], Q-Fine2000, 3000 [Tonda Pharmaceutical Co., Ltd.], Synthetic zeolite A-4, Synthetic zeolite F-9 [Toyo Soda Industry Co., Ltd.] ], Natural zeolite [Nippon Active Soil Co., Ltd.], bentonite [Wako Pure Chemical Industries, Ltd.], etc. can be used, but are not limited thereto.
ヒアルロン酸類含有液（発酵液も含む）の活性炭及び無機吸着剤処理を行うにあたり、含有液のｐＨは３〜１２、特に４〜１１が好ましい。温度は０〜８０℃、ヒアルロン酸類濃度は０．１〜２０ｇ／ｌ、好ましくは１〜１０ｇ／ｌである。含有液のｐＨが２．５未満の場合にはヒアルロン酸類のゲル化が生じ、１２以上の場合には、ヒアルロン酸が徐々に分解し、分子量が低下する。温度が８０℃を越えると処理中にヒアルロン酸類が徐々に分解し、分子量が低下する。ヒアルロン酸類濃度が１０ｇ／ｌを越えると溶液粘度が高くなり活性炭及び／又は無機吸着剤処理が難しくなる。ヒアルロン酸類含有液の粘度を下げる目的で食塩等の塩を共存させてもよい。 In performing the activated carbon and inorganic adsorbent treatment of the hyaluronic acid-containing liquid (including the fermented liquid), the pH of the containing liquid is preferably 3 to 12, particularly 4 to 11. The temperature is 0 to 80 ° C., and the hyaluronic acid concentration is 0.1 to 20 g / l, preferably 1 to 10 g / l. When the pH of the containing liquid is less than 2.5, gelation of hyaluronic acid occurs, and when it is 12 or more, hyaluronic acid is gradually decomposed and the molecular weight is lowered. When the temperature exceeds 80 ° C., hyaluronic acids are gradually decomposed during the treatment, and the molecular weight is lowered. When the hyaluronic acid concentration exceeds 10 g / l, the solution viscosity becomes high, and the activated carbon and / or inorganic adsorbent treatment becomes difficult. For the purpose of lowering the viscosity of the hyaluronic acid-containing liquid, a salt such as sodium chloride may coexist.
本工程の処理方法として、通常、活性炭及び無機吸着剤又は無機吸着剤のみをヒアルロン酸類含有液（発酵液も含む）に１回懸濁・撹拌処理するだけで充分であるが、好ましくは、無機吸着剤、活性炭の順で別々に懸濁・撹拌処理した方がよい。 As a treatment method in this step, it is usually sufficient to suspend and agitate only activated carbon and an inorganic adsorbent or an inorganic adsorbent once in a hyaluronic acid-containing liquid (including a fermented liquid). It is better to suspend and stir the adsorbent and activated carbon separately in this order.
ヒアルロン酸類含有液を処理する活性炭の量は、ヒアルロン酸類含有液１００重量％に対し０．５〜２０．０重量％、好ましくは５．０〜２０．０重量％である。無機吸着剤の量は、ヒアルロン酸類含有液１００重量％に対し０．５〜２０．０重量％、好ましくは５．０〜２０．０重量％である。撹拌時間は１０分〜２時間、好ましくは２０分〜１時間である。 The amount of the activated carbon for treating the hyaluronic acid-containing liquid is 0.5 to 20.0% by weight, preferably 5.0 to 20.0% by weight, with respect to 100% by weight of the hyaluronic acid-containing liquid. The amount of the inorganic adsorbent is 0.5 to 20.0% by weight, preferably 5.0 to 20.0% by weight, based on 100% by weight of the hyaluronic acid-containing liquid. The stirring time is 10 minutes to 2 hours, preferably 20 minutes to 1 hour.
Ｂ工程： ヒアルロン酸類含有液を酸性側のｐＨ範囲内に調整した後、限外ろ過膜にて透析処理することにより、蛋白質、乳酸、金属、培地由来の無機塩類等を除去する工程。 Step B: A step of removing protein, lactic acid, metal, medium-derived inorganic salts, and the like by dialysis with an ultrafiltration membrane after adjusting the hyaluronic acid-containing liquid within the acidic pH range.
本工程に用いる限外ろ過膜は、酢酸セルロース、ポリアクリロニトリル、ポリスルフォン、ポリエーテルスルフォン、ハロゲン化ポリオレフィン、ポリアミド、ポリフッ化ビニリデン等が挙げられるが、これらに制限されるものではない。又、限外ろ過膜の材質や構造等にとらわれるものではない。ろ過形状としては、平面膜、管型、スパイラル、ホロファイバー等、どのようなモジュール形式でもよい。 Examples of the ultrafiltration membrane used in this step include, but are not limited to, cellulose acetate, polyacrylonitrile, polysulfone, polyether sulfone, halogenated polyolefin, polyamide, and polyvinylidene fluoride. Further, it is not limited by the material or structure of the ultrafiltration membrane. The filtration shape may be any module type such as a flat membrane, a tube shape, a spiral, or a holofiber.
限外ろ過膜のろ過精度は、分画分子量は１〜１０万の範囲で使用できる。例えば、ＰＭ―１０、ＰＭ―５０、ＰＭ―１００（ロミコン社製）、ＮＴＵ―３０５０（日東電工社製）、ＩＲＩＳ３０６５（ローヌ・プラン社製）、ＦＳ―１００（旭化成社製）、ＤＵＳＯ４００（ダイセル化学工業社製）、ＭＵ―６０２２、ＭＵ―６３０２、ＭＵ―６３０３（クラレ社製）等が使用できるが、これらに限定されるものではない。 As for the filtration accuracy of the ultrafiltration membrane, the molecular weight cut off can be used in the range of 1 to 100,000. For example, PM-10, PM-50, PM-100 (Romicon), NTU-3050 (Nitto Denko), IRIS 3065 (Rhone Plan), FS-100 (Asahi Kasei), DUSO400 (Daicel) Chemical Industry Co., Ltd.), MU-6022, MU-6302, MU-6303 (Kuraray Co., Ltd.) and the like can be used, but are not limited thereto.
ヒアルロン酸類含有液の限外ろ過膜透析処理を行うにあたり、含有液のｐＨは２．５〜５．０、温度は０〜８０℃、ヒアルロン酸類濃度は０．１〜１０ｇ／ｌが好ましい。含有液のｐＨが２．５未満又は温度が８０℃を越す場合には、ヒアルロン酸が徐々に分解し、分子量が低下する。ヒアルロン酸類濃度が１０ｇ／ｌを越えると溶液粘度が高くなり限外ろ過処理が難しくなり、ヒアルロン酸類含有液の粘度を下げる目的で食塩等の塩を共存させてもよい。 In performing the ultrafiltration membrane dialysis treatment of the hyaluronic acid-containing liquid, the pH of the containing liquid is preferably 2.5 to 5.0, the temperature is 0 to 80 ° C., and the hyaluronic acid concentration is preferably 0.1 to 10 g / l. When the pH of the containing liquid is less than 2.5 or the temperature exceeds 80 ° C., hyaluronic acid is gradually decomposed to lower the molecular weight. When the hyaluronic acid concentration exceeds 10 g / l, the solution viscosity becomes high and the ultrafiltration treatment becomes difficult, and a salt such as sodium chloride may be coexisted for the purpose of reducing the viscosity of the hyaluronic acid-containing liquid.
本工程の処理方法としては、一般的な定容量に純水を希釈しながら透析ろ過する方法あるいは、濃縮と純水の希釈を繰り返しながら透析ろ過する方法がある。この時の透析ろ過は全量ろ過法または、クロスフロー法のいずれでもよいが、好ましくはクロスフロー法である。 As a processing method in this step, there is a method of diafiltration while diluting pure water to a general constant volume, or a method of diafiltration while repeating concentration and dilution of pure water. The diafiltration at this time may be either a total filtration method or a cross flow method, but preferably a cross flow method.
ヒアルロン酸類含有液を限外ろ過透析処理を行う場合、クロスフローに通液する濃縮側の線速度はヒアルロン酸類含有液の性状や限外ろ過膜の種類により異なり一律に規定する事ができないが、例えば、中空糸の場合５ｍ／ｓ以下が好ましい。線速度が５ｍ／ｓ以上の場合には、せん断によりヒアルロン酸の分子量の低下をきたす。又、限外ろ過透析処理を行うにあたり、膜前処理として、２％以下のアルカリ（例えば、水酸化ナトリウム水溶液）、過酸化物（例えば、次亜塩素酸ナトリウム水溶液）、界面活性剤、クエン酸、クエン酸アンモニウム、酵素洗剤等の薬剤で膜を洗浄処理する化学的方法や、フラッシング、スポンジボール、空気インジェクション法等の物理的方法により行うことが望ましい。 When the hyaluronic acid containing liquid is subjected to ultrafiltration dialysis treatment, the linear velocity on the concentration side passing through the cross flow varies depending on the properties of the hyaluronic acid containing liquid and the type of the ultrafiltration membrane, but cannot be specified uniformly. For example, in the case of a hollow fiber, 5 m / s or less is preferable. When the linear velocity is 5 m / s or more, the molecular weight of hyaluronic acid is reduced by shearing. In addition, in performing ultrafiltration dialysis treatment, as membrane pretreatment, 2% or less of alkali (for example, sodium hydroxide aqueous solution), peroxide (for example, sodium hypochlorite aqueous solution), surfactant, citric acid It is desirable to use a chemical method of washing the membrane with a chemical such as ammonium citrate or an enzyme detergent, or a physical method such as flushing, sponge ball, or air injection.
Ｃ工程： ヒアルロン酸類含有液をセラミック膜からなる濾材に接触させることにより、微粒子、発熱性物質やタンパク質等を効率良く除去し、更に使用したセラミック膜は通常の洗浄処理で容易に性能が回復し、高純度のヒアルロン酸類を取得する工程。 Step C: By contacting the hyaluronic acid-containing liquid with a filter medium made of a ceramic membrane, fine particles, pyrogens, proteins, etc. are efficiently removed, and the performance of the used ceramic membrane can be easily recovered by ordinary washing treatment. The process of obtaining high purity hyaluronic acids.
本工程に用いるセラミック膜はＡｌ２Ｏ３、ＺｒＯ３、ＳｉＯ２、ＴｉＯ２、Ｓｉ３Ｎ４、ＳｉＣ、ＢＮ、ＭｇＯ、及びＺｎＯ等の適当な配合比により焼結させた物が挙げられるが、これらに制限されるものではない。ろ過形状としては、平面膜、管型、カートリッジ、ホロファイバー等、どのような形式でもよい。 Examples of the ceramic film used in this step include those sintered at an appropriate mixing ratio such as Al 2 O 3 , ZrO 3 , SiO 2 , TiO 2 , Si 3 N 4 , SiC, BN, MgO, and ZnO. However, it is not limited to these. The filtration shape may be any type such as a flat membrane, a tube type, a cartridge, a holofiber, and the like.
セラミック膜のろ過精度は０．１μｍ〜１０μｍの範囲で使用できるが、微粒子の除去を目的とする場合には２μｍ以下、ウイルスやバクテリアの完全除去を目的とする場合には０．４５以下のろ過精度が好ましい。 The filtration accuracy of the ceramic membrane can be used in the range of 0.1 μm to 10 μm, but it is 2 μm or less for the purpose of removing fine particles, and 0.45 or less for the purpose of complete removal of viruses and bacteria. Accuracy is preferred.
セラミック膜として、例えば、ＮＧＫ モノリス型セラミック膜フィルター、ＮＧＫ チューブラー型セラミック膜フィルター（日本ガイシ社製）、セラミックフィルターＣＥＬＡＦＬＯ（日本ミリポア・リミテッド社製）等が使用できるが、これらに限定されるものではない。 As the ceramic membrane, for example, NGK monolithic ceramic membrane filter, NGK tubular ceramic membrane filter (manufactured by NGK Corporation), ceramic filter CELAFLO (manufactured by Nippon Millipore Limited), etc. can be used, but are not limited thereto. is not.
ヒアルロン酸類含有液のセラミック膜処理を行うにあたり、含有液のｐＨは３〜１０、温度は０〜８０℃、ヒアルロン酸類濃度は０．１〜１０ｇ／ｌが好ましい。含有液のｐＨが３未満の場合にはヒアルロン酸類のゲル化が生じ、温度が８０℃を越す場合には、ヒアルロン酸が徐々に分解し、分子量が低下する。ヒアルロン酸類濃度が１０ｇ／ｌを越えると溶液粘度が高くなりセラミック膜処理が難しくなり、ヒアルロン酸類含有液の粘度を下げる目的で食塩等の塩を共存させてもよい。 In performing the ceramic membrane treatment of the hyaluronic acid-containing liquid, the pH of the containing liquid is preferably 3 to 10, the temperature is 0 to 80 ° C., and the hyaluronic acid concentration is preferably 0.1 to 10 g / l. When the pH of the containing liquid is less than 3, gelation of hyaluronic acid occurs, and when the temperature exceeds 80 ° C., hyaluronic acid gradually decomposes and the molecular weight decreases. When the hyaluronic acid concentration exceeds 10 g / l, the solution viscosity becomes high and ceramic membrane treatment becomes difficult, and a salt such as sodium chloride may be coexisted for the purpose of reducing the viscosity of the hyaluronic acid-containing solution.
本工程の処理方法としては、クロスフローや直ろ過方法が使用できる。通常、セラミック膜にヒアルロン酸類含有液を１図通液するだけで充分であるが、必要なら、反復処理しても良い。また、セラミック膜は１段、あるいは複数個を直列に接続して使用することもできる。 As a processing method of this process, a cross flow or a direct filtration method can be used. Usually, it is sufficient to pass a hyaluronic acid-containing solution through the ceramic membrane for one figure, but if necessary, it may be repeated. The ceramic film can be used in a single stage or a plurality of ceramic films connected in series.
ヒアルロン酸類含有液をセラミック膜に通液する速度はヒアルロン酸類含有液の性状や膜の種類によりことなり一律に規定することが出来ないが、有効ろ過面積１ｍ２当たり４０ｌ／ｈｒを越えると閉塞を生じやすくなる。その結果、ゲル付着等により収率低下となるので、有効ろ過面積１ｍ２当たり２００ｌ／ｈｒ以下が好ましい。 The rate at which the hyaluronic acid-containing liquid is passed through the ceramic membrane cannot be uniformly defined depending on the properties of the hyaluronic acid-containing liquid and the type of the membrane, but if it exceeds 40 l / hr per 1 m 2 of effective filtration area, it will block. It tends to occur. As a result, the yield decreases due to gel adhesion or the like, and therefore, 200 l / hr or less per 1 m 2 of effective filtration area is preferable.
微粒子、タンパク質や発熱性物質等の不純物を吸着する能力が低下したセラミック膜は、通常の酸、アルカリ洗浄、酵素洗浄、界面活性剤洗浄、キレート洗浄、次亜塩素酸洗浄、過酸化水素洗浄等の化学的洗浄、フラッシングや逆洗浄の物理的洗浄等で初期性能に回復させることが出来る。 Ceramic films with reduced ability to adsorb impurities such as fine particles, proteins and pyrogens are usually used for acid, alkali cleaning, enzyme cleaning, surfactant cleaning, chelate cleaning, hypochlorous acid cleaning, hydrogen peroxide cleaning, etc. The initial performance can be restored by chemical cleaning, flushing or physical cleaning such as reverse cleaning.
Ｄ工程：ヒアルロン酸類含有液を天然物を基材とするクロマトグラフィー用充填剤又は、担体に鎖長が炭素数８以上の置換又は未置換の炭化水素基を結合させた逆相クロマトグラフィー用充填剤に接触させ、発熱性物質、タンパク質、核酸、金属等の不純物を効率よく除去し、高純度のヒアルロン酸類を取得する工程。 Step D: Packing for chromatography using a hyaluronic acid-containing liquid as a base material for a natural product or reverse phase chromatography in which a substituted or unsubstituted hydrocarbon group having a chain length of 8 or more carbon atoms is bound to a carrier A process of obtaining high-purity hyaluronic acids by efficiently removing impurities such as pyrogens, proteins, nucleic acids, metals, etc. by contacting with an agent.
本工程に用いられる天然物を基材とするクロマトグラフィー用充填剤の担体は、セルロース、ヒスチジン、メチルグルタメート、キチンキトサン系の球状又は破砕状のものが好ましい。逆相クロマトグラフィー用充填剤の担体は、シリカゲル又は、合成樹脂の球状又は破砕状のものが好ましい。その平均粒径は、数μｍから約１００μｍのものが好ましい。 The carrier for the chromatographic filler based on natural products used in this step is preferably cellulose, histidine, methylglutamate, chitin chitosan-based spherical or crushed. The carrier for the packing for reverse phase chromatography is preferably silica gel or spherical or crushed synthetic resin. The average particle diameter is preferably from several μm to about 100 μm.
本工程に用いられる逆相クロマトグラフィー用充填剤は、担体に、置換又は未置換の炭化水素基を結合させたものであり、特に鎖長が炭素数８以上のものを用いることを特徴とする。その例として、オクチル基、オクタデシル基およびこれらの末端を改変した官能基等が挙げられるが、これらに制限されるものではない。 The packing material for reverse phase chromatography used in this step is obtained by bonding a substituted or unsubstituted hydrocarbon group to a carrier, and particularly having a chain length of 8 or more. . Examples thereof include, but are not limited to, an octyl group, an octadecyl group, and a functional group obtained by modifying these ends.
鎖長が炭素数８以下の置換又は未置換の炭化水素基として、例えばトリメチル基、ブチル基、フェニル基、シアノプロピル基、アミノプロピル基、等が挙げられるが、これらは本工程には適さない。 Examples of the substituted or unsubstituted hydrocarbon group having a chain length of 8 or less carbon atoms include a trimethyl group, a butyl group, a phenyl group, a cyanopropyl group, and an aminopropyl group, but these are not suitable for this step. .
本工程に適するものとして、例えば市販されているオクチル基をシリカゲルに化学的に結合させた逆相クロマトグラフィー用充填剤［ＹＭＣ・ＧEＬ Ｃ８−１２０−Ｓ５（ＹＭＣ製）］、［ＹＭＣ・ＧEＬ Ｃ８−３００−Ｓ５（ＹＭＣ製）］、オクタデシル基をシリカゲルに化学的に結合させた逆相クロマトグラフィー用充填剤［ＹＭＣ・ＧEＬ ＯＤＳ−ＡＭ−１２０−Ｓ５０（ＹＭＣ製）］、［ＹＭＣ・ＧＥＬ ＯＤＳ−Ａ１２０−Ｓ３０／５０（ＹＭＣ製）］、［ＹＭＣ・ＧＥＬ ＯＤＳ−ＡＱ６０−Ｓ５０（ＹＭＣ製）］、［ＹＭＣ・ＧＥＬ ＯＤＳ−ＡＱ１２０−Ｓ５０（ＹＭＣ製）］、アルキル基をビニルアルコール・コポリマーに化学的に結合させた逆相クロマトグラフィー用充填剤［ＯＤＰ−４００（昭和電工社製）］、［Ｃ８Ｐ−４００（昭和電工社製）］、［Ｏｃｔａｄｅｃｙｌ−４ＰＷ（東ソー社製）］、γ−メチルＬ−グルタメートをアミノ化した［パイロトール（日本濾水機社製）］、セルロースをアミノ化した［パイロセップ−Ｃ（ダイセル化学社製）］、キチンキトサンをアミノ化した［クリムーバ（栗田工業社製）］などが挙げられるが、これらに制限されるものではない。 As suitable for this step, for example, a filler for reverse phase chromatography in which a commercially available octyl group is chemically bonded to silica gel [YMC · GEL C8-120-S5 (manufactured by YMC)], [YMC · GEL C8 -300-S5 (manufactured by YMC)], filler for reverse phase chromatography in which octadecyl group is chemically bonded to silica gel [YMC · GEL ODS-AM-120-S50 (manufactured by YMC)], [YMC · GEL ODS -A120-S30 / 50 (manufactured by YMC)], [YMC · GEL ODS-AQ60-S50 (manufactured by YMC)], [YMC · GEL ODS-AQ120-S50 (manufactured by YMC)], and an alkyl group into a vinyl alcohol copolymer. Chemically bonded filler for reverse phase chromatography [ODP-400 (manufactured by Showa Denko)], [C8 -400 (manufactured by Showa Denko KK)], [Octadecyl-4PW (manufactured by Tosoh Corporation)], [pyrotol (manufactured by Nippon Seiki Co., Ltd.)] aminated γ-methyl L-glutamate, and [a pyrosep aminated cellulose] -C (manufactured by Daicel Chemical Industries, Ltd.)] and [Crimova (manufactured by Kurita Kogyo Co., Ltd.)] obtained by amination of chitin chitosan, but are not limited thereto.
天然物を基材とするクロマトグラフィー用充填剤又は、逆相クロマトグラフィー用充填剤に接触させる溶媒は、水−有機溶媒の混合溶媒系に塩を含むものが一般的に用いられるが、本工程に於いては、塩を含むヒアルロン酸類水溶液を天然物を基材とするクロマトグラフィー用充填剤又は、逆相クロマトグラフィー用充填剤に接触させることが特に良い。本工程に用いられる塩の種類は、硫酸アンモニウム、クエン酸ナトリウム、クエン酸力リウム、リン酸カリウム、リン酸ナトリウム、硫酸ナトリウム、硫酸カリウム、硫酸マグネシウム、塩化ナトリウム、塩化カリウム等を使用することが出来るが、これに制限されるものではない。すなわち、ヒアルロン酸類含有液を天然物を基材とするクロマトグラフィー用充填剤又は、逆相クロマトグラフィー用充填剤に接触させるに当たり、ヒアルロン酸類含有液は、水系で、塩濃度は０．５〜２０ｗ／ｖ％が好ましい。また水溶液のｐＨは３〜１２、温度は０〜８０℃、ヒアルロン酸類の濃度は０．１〜１０ｇ／ｌが好ましい。 As a solvent to be brought into contact with a packing material for chromatography based on a natural product or a packing material for reverse phase chromatography, a solvent containing a salt in a mixed solvent system of water-organic solvent is generally used. In this case, it is particularly preferable that the aqueous solution of hyaluronic acid containing a salt is brought into contact with a packing material for chromatography based on a natural product or a packing material for reverse phase chromatography. As the type of salt used in this step, ammonium sulfate, sodium citrate, potassium citrate, potassium phosphate, sodium phosphate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium chloride, potassium chloride and the like can be used. However, it is not limited to this. That is, when the hyaluronic acid-containing liquid is brought into contact with the packing material for chromatography based on natural products or the packing material for reverse phase chromatography, the hyaluronic acid-containing liquid is an aqueous system and the salt concentration is 0.5 to 20 w. / V% is preferred. The pH of the aqueous solution is preferably 3 to 12, the temperature is 0 to 80 ° C., and the concentration of hyaluronic acid is preferably 0.1 to 10 g / l.
天然物を基材とするクロマトグラフィー用充填剤又は逆相クロマトグラフィー用充填剤に接触させる方法としては、ヒアルロン酸類含有液に逆相クロマトグラフィー用充填剤を添加して、バッチ式で撹拌する方法と、充填塔等に充填後、ヒアルロン酸類含有液を通液処理する方法、またはその組合せや反復も可能であるが、通常は、処理条件の選択により、１回の処理で十分である。 As a method of bringing into contact with a packing material for chromatography based on a natural product or a packing material for reverse phase chromatography, a method of adding a packing material for reverse phase chromatography to a hyaluronic acid-containing liquid and stirring in a batch system A method of passing the hyaluronic acid-containing liquid through the packed tower or the like, or a combination or repetition thereof, can be used, but usually one treatment is sufficient depending on selection of treatment conditions.
発熱性物質、タンパク質、核酸等の不純物を除去する能力の低下した天然物を基材としたクロマトグラフィー用充填剤又は、逆相クロマトグラフィー用充填剤は、通常の脱着、洗浄、再生処理で簡単に初期の性能に回復させることが出来る。 Chromatographic packing material or reverse phase chromatography packing material based on natural products with reduced ability to remove pyrogens, proteins, nucleic acids and other impurities can be easily removed by ordinary desorption, washing and regeneration processes. The initial performance can be recovered.
Ｅ工程： ヒアルロン酸類含有液を親水性膜、ポリアミド膜、疎水性膜、プラスのゼータ電位を有する膜から選ばれた一種の濾材に接触させることにより、発熱性物質やタンパク質等を効率よく吸着除去し、高純度のヒアルロン酸類を取得する工程。 Step E: By bringing the hyaluronic acid-containing liquid into contact with a kind of filter medium selected from a hydrophilic membrane, a polyamide membrane, a hydrophobic membrane, and a membrane having a positive zeta potential, the exothermic substances and proteins are efficiently adsorbed and removed. And obtaining high-purity hyaluronic acid.
本工程に用いる親水性膜としては、ポリスルフォン等であり、ポリアミド膜としては、６−ナイロン、６，６−ナイロン、７−ナイロン、１１−ナイロン、１２−ナイロン、６，１１−ナイロン、６，１２−ナイロン等であり、疎水性膜としては、ポリビニリデンジフルオライド、ポリテトラフロロエチレン、ポリエチレン、ポリプロピレン、等であり、これらに制限されるものではない。また、荷電膜としては、ヒアルロン酸類含有水溶液中でゼータ電位がプラスに荷電している膜であれば良く、例えば、膜の材質や構造、荷電方法などにとらわれるものではない。 The hydrophilic membrane used in this step is polysulfone or the like, and the polyamide membrane is 6-nylon, 6,6-nylon, 7-nylon, 11-nylon, 12-nylon, 6,11-nylon, 6 , 12-nylon, etc., and the hydrophobic membrane is polyvinylidene difluoride, polytetrafluoroethylene, polyethylene, polypropylene, etc., but is not limited thereto. The charged film may be any film that has a positive zeta potential in a hyaluronic acid-containing aqueous solution, and is not limited to, for example, the material or structure of the film, or the charging method.
また、膜の材質や構造等にとらわれるものではない。濾材構造として表面濾過構造や深層濾過構造等が使用できる。また、形状はシートやカートリッジ、ホロファイバー等、どのような形でも良い。 Further, it is not restricted by the material or structure of the film. A surface filtration structure, a depth filtration structure, or the like can be used as the filter medium structure. Further, the shape may be any shape such as a sheet, a cartridge, or a holofiber.
膜の濾過精度は０．１μｍ〜１０μｍの範囲で使用できるが、ウイルスやバクテリアの完全除去を目的とする場合には０．４５μｍ以下の濾過精度が好ましい。 Although the filtration accuracy of the membrane can be used in the range of 0.1 μm to 10 μm, the filtration accuracy of 0.45 μm or less is preferable for the purpose of complete removal of viruses and bacteria.
具体的には、親水性膜としては例えば、デュラポア（日本ミリポア社製）、スーポア（ゲルマンサイエンス社製）、ポリアミド膜としては例えば、ウルチポアＮ６６、ポジダイン（以上、日本ポール社製）、ゼータポア（以上、キュノ社製）。 Specifically, as a hydrophilic membrane, for example, Durapore (manufactured by Nihon Millipore), Supor (manufactured by Gelman Science), and as a polyamide membrane, for example, Ultipor N66, Posodyne (manufactured by Nihon Pall), Zetapore (and above) , Manufactured by Cuno).
疎水性膜としては、例えばデュラポア（日本ミリポア社製）、エンフロン（日本ポーラ社製）、マイクロフローフィルター（キュノ社製）、ステラポア（三菱レーヨン社製）。 Examples of the hydrophobic membrane include Durapore (manufactured by Nippon Millipore), Enflon (manufactured by Nippon Pola), microflow filter (manufactured by Cuno), and Sterapore (manufactured by Mitsubishi Rayon).
荷電膜として、例えばポジダイン、プロファイル プラス、ウルチポアＧＦ ＰＬＵＳ （以上、日本ポール社製）、ゼータポア、ゼータプラス（以上、キュノ社製）等が使用できるがこれらに制限されるものではない。 Examples of the charged membrane include, but are not limited to, Posodyne, Profile Plus, Ultipore GF PLUS (manufactured by Nippon Pole), Zetapore, Zetaplus (manufactured by Cuno), and the like.
ヒアルロン酸類含有液の膜処理を行うに当たり、含有液のｐＨは３〜１２、特に４〜９、温度は０〜８０℃、ヒアルロン酸類濃度は０．１〜１０ｇ／ｌが良い。含有液のｐＨが２．５未満の場合にはヒアルロン酸類のゲル化が生じ、ろ過困難となる。ｐＨが９を越えた場合には、膜の吸着効果が低下する傾向にある。 In performing the membrane treatment of the hyaluronic acid-containing liquid, the pH of the containing liquid is preferably 3 to 12, particularly 4 to 9, the temperature is 0 to 80 ° C., and the hyaluronic acid concentration is 0.1 to 10 g / l. When the pH of the containing liquid is less than 2.5, hyaluronic acid gelation occurs and filtration becomes difficult. When the pH exceeds 9, the membrane adsorption effect tends to decrease.
温度が８０℃を越えると処理中にヒアルロン酸類が徐々に分解し、分子量が低下する。 When the temperature exceeds 80 ° C., hyaluronic acids are gradually decomposed during the treatment, and the molecular weight is lowered.
ヒアルロン酸類濃度が１０ｇ／ｌを越えると溶液粘度が高くなり膜処理が難しくなる。 When the hyaluronic acid concentration exceeds 10 g / l, the solution viscosity becomes high and the membrane treatment becomes difficult.
ヒアルロン酸類水溶液の粘度を下げる目的で食塩等の塩を共存させても良いが、高濃度になるとポリアミド膜の吸着効果が低下する傾向にある。 For the purpose of lowering the viscosity of the aqueous hyaluronic acid solution, a salt such as sodium chloride may be coexistent, but when the concentration becomes high, the adsorption effect of the polyamide membrane tends to decrease.
本発明の処理方法としては、通常、膜にヒアルロン酸類水溶液を１回通液するだけで充分であるが、必要なら、反復処理しても良い。また、膜は１段、あるいは複数個を直列に接続して使用することもできる。 In the treatment method of the present invention, it is usually sufficient to pass the hyaluronic acid aqueous solution once through the membrane. However, if necessary, the treatment may be repeated. Further, the film can be used in one stage or a plurality of films connected in series.
ヒアルロン酸類含有液を膜に通液する速度はヒアルロン酸類含有液の性状や膜の種類により異なり一律に規定することが出来ないが、有効ろ過面積１ｍ２当たり５００ｌ／ｈｒを越えると閉塞を生じやすくなる。その結果、ゲル付着等により収率低下となるので、有効ろ過面積１ｍ２当たり５００ｌ／ｈｒ以下が好ましい。 Rate of passed through hyaluronic acid-containing liquid film is not able to define uniformly depends on the type of properties and film of hyaluronic acid-containing liquid, tends to occur clogging exceeds the effective filtration area 1 m 2 per 500 l / hr Become. As a result, the yield decreases due to gel adhesion or the like, and therefore, 500 l / hr or less per 1 m 2 of effective filtration area is preferable.
ただし、疎水性膜処理を行うにあたり、膜前処理として、エタノールを通液し親水化処理することが望ましい。タンパク質や発熱性物質等の不純物を吸着する能力が低下した膜は、通常の酸・アルカリ洗浄、酵素洗剤、界面活性剤洗浄、キレート洗浄、次亜塩素酸洗浄、過酸化水素洗浄等の化学的洗浄、フラッシングや逆洗等の物理的洗浄等で初期の性能に回復させることが出来る。 However, in carrying out the hydrophobic membrane treatment, it is desirable that the membrane is pretreated by passing ethanol through it to make it hydrophilic. Membranes with reduced ability to adsorb impurities such as proteins and pyrogens are treated with chemicals such as normal acid / alkali cleaning, enzyme detergent, surfactant cleaning, chelate cleaning, hypochlorous acid cleaning, and hydrogen peroxide cleaning. The initial performance can be restored by physical washing such as washing, flushing and back washing.
以上、上記は、Ａ、Ｂ、Ｃ、Ｄ、Ｅの工程順に処理することを詳細に説明したが、工程順は請求項２〜９のような工程順に処理することも可能であるが、特に好ましくはＡ、Ｂ、Ｃ、Ｄ、Ｅの工程順に処理することである。 As mentioned above, although it processed in order of the process order of A, B, C, D, and E in detail, the process order can also be processed in the order of processes as in claims 2 to 9, Preferably, the treatment is performed in the order of steps A, B, C, D, and E.
以下、本発明を実施例により更に具体的に説明するが、本発明はこれらに限定されない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these.
実施例１
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤として活性白土［日本活性白土社製］６．７５０ｋｇ（１５重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ―６３０３―ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 1
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. 6.750 kg (15% by weight) of activated clay (manufactured by Nippon Kakuhakudo Co., Ltd.) was added, and suspended and stirred for 0.5 hours at 50 rpm using a stirrer. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-fold pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤として活性白土［日本活性白土社製］３．３７５ｋｇ（７．５重量％）、有機吸着剤としてクリムーバ［栗田工業社製］３．３７５ｋｇ（７．５重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４２ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 2
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Activated clay (made by Nippon Activated Shiraito Co., Ltd.) 3.375 kg (7.5% by weight), and Crimova [made by Kurita Kogyo Co., Ltd.] 3.375 kg (7.5% by weight) as an organic adsorbent were added, and 50 rpm was used using a stirrer. The suspension was stirred for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 142 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］１．１２５５ｋｇ（２．５重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．３７５ｋｇ（７．５重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 3
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 1.1255 kg (2.5 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.375 kg (7.5 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例４
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてＱ−Ｆｉｎｅ２０００［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．６００ｋｇ（８．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１３９ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 4
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Q-Fine2000 [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%), Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.600 kg (8.0 wt%) as activated carbon was added, and 50 rpm was used using a stirrer. The suspension was stirred for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, it is effectively filtered through a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 hole, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 139 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例５
［Ａ工程］
ストレプトコッカス・エクイＦＭ―１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し有機吸着剤としてＰＭＬＧ―ＥＤ［ファルマシア社製］１．１２５ｋｇ（２．５重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］１．１２５ｋｇ（２．５重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター（日本ポール社製）に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 5
[Step A]
45 l of fermented liquid (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultured using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an organic adsorbent. PMLG-ED (Pharmacia Co., Ltd.) 1.125 kg (2.5 wt%) and Shirakaba P (Takeda Pharmaceutical Co., Ltd.) 1.125 kg (2.5 wt%) as activated carbon were added and stirred at 50 rpm. The suspension was stirred for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例６
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤として活性白土［日本活性白土社製］２．２５０ｋｇ（５．０重量％）、活性炭としてウエットな白鷺Ａ［武田薬品工業社製］２．２５０ｋｇ（５．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０１を線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 6
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. 2.250 kg (5.0 wt%) of activated clay (made by Nippon Katsuhaku Co., Ltd.) and 2.250 kg (5.0 wt%) of wet white rice cake A (made by Takeda Pharmaceutical Company Limited) as activated carbon were added, and the stirrer was added. Using suspension at 50 rpm for 0.5 hour, the suspension was stirred. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and the solution 701 obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例７
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤として合成ゼオライトＡ−４［東洋曹達社製］０．２２５ｋｇ（０．５重量％）、活性炭としてウエットな白鷺Ａ［武田薬品工業社製］２．０２５ｋｇ（４．５重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 7
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Synthetic zeolite A-4 [manufactured by Toyo Soda Co., Ltd.] 0.225 kg (0.5 wt%) and wet white birch A [manufactured by Takeda Pharmaceutical Company Limited] 2.025 kg (4.5 wt%) as activated carbon were added and stirred. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, it is effectively filtered through a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 hole, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例８
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 8
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 g of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 hole, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例９
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤として活性白土［日本活性白土社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 9
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Activated white clay [manufactured by Nippon Active White Soil Co., Ltd.] 0.450 kg (1.0% by weight), activated carbon Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0% by weight) was added, and 50 rpm was used using a stirrer. The suspension was stirred for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１０
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤として合成ゼオライトＡ−４［東洋曹達工業社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 10
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Synthetic zeolite A-4 [manufactured by Toyo Soda Kogyo Co., Ltd.] 0.450 kg (1.0 wt%), and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer Was suspended and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. Equilibrated in the order of sodium solution, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr [step E].
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１１
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてベントナイト［和光純薬工業社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 11
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Bentonite [manufactured by Wako Pure Chemical Industries, Ltd.] 0.450 kg (1.0 wt%), Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon was added, and 50 rpm was used using a stirrer. The suspension was stirred for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１２
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 12
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank, and white birch as activated carbon P [made by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) was added, and suspended and stirred for 0.5 hours at 50 rpm using a stirrer. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１３
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ２．９に調整した後、分画分子量５万材質ポリスルフォンの限外ろ過膜ＰＭ―５０［ロミコン社製］２ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４３ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 13
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 2.9, a linear flow rate of 0.5 m / s at 2 m 2 of ultrafiltration membrane PM-50 (manufactured by Romicon) with a molecular weight cut off of 50,000 and a polysulfone material. Then, the volume ratio was concentrated twice and the dilution with the same volume of pure water was repeatedly performed with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, it is effectively filtered into a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 hole, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 143 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１４
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ２．９に調整した後、分画分子量１０万材質ポリスルフォンの限外ろ過膜ＰＭ―１００［ロミコン社製］２ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４３ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 14
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in the step A to pH 2.9, a circulation linear velocity of 0.5 m / s at 2 m 2 of ultrafiltration membrane PM-100 (manufactured by Romicon) of polysulfone with a fractional molecular weight of 100,000 material Then, the volume ratio was concentrated twice and the dilution with the same volume of pure water was repeatedly performed with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, it is effectively filtered into a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 hole, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 143 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１５
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ２．９に調整した後、分画分子量１万材質ポリスルフォンの限外ろ過膜ＰＭ−１０［ロミコン社製］２ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１３９ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 15
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in the step A to pH 2.9, a linear molecular velocity of 0.5 m / s at an ultrafiltration membrane PM-10 made of polysulfone with a fractional molecular weight of 10,000 material (Romicon) 2 m 2 Then, the volume ratio was concentrated twice and the dilution with the same volume of pure water was repeatedly performed with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 139 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１６
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ２．９に調整した後、分画分子量３万材質ポリエーテルスルフォンの限外ろ過膜ＦＳ―１００［旭化成社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 16
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in the step A to pH 2.9, a circulation linear velocity of 0.5 m / min was obtained at 5 m 2 of an ultrafiltration membrane FS-100 (manufactured by Asahi Kasei Co., Ltd.) having a molecular weight cut off of 30,000. s, volume ratio 2 times concentration, and equal volume pure water dilution were repeatedly treated with dialysis 11 times.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１７
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微王研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４―２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ２．９に調整した後、分画分子量２万材質ポリスルフォンの限外ろ過膜ＮＴＵ―３０５０［日東電工社製］３ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 17
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Mioh Kenhyo 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting the solution 45l obtained in the Process A to pH 2.9, fractionation molecular weight of 20,000 Material polysulfone ultrafiltration membrane NTU-3050 at [manufactured by Nitto Denko Corporation] 3m 2 circulation linear velocity 0.5 m / s, volume ratio 2 times concentration, and equal volume pure water dilution were repeatedly treated with dialysis 11 times.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 2.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１８
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ２．９に調整した後、分画分子量４万材質ポリビニリデンジフロライドの限外ろ過膜ＩＲＩＳ−３０６５［ローヌプラン社製］３ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０ｘ１０００ｍｍ、φ４ｘ１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４０ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 18
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in the step A to pH 2.9, the circulation linear velocity was 0 at 3 m 2 of an ultrafiltration membrane IRIS-3065 (Rhone Plan) made of polyvinylidene difluoride with a molecular weight cut off of 40,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm. The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed with 8 l of ethanol three times, and vacuum dried at 40 ° C. to obtain 140 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例１９
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ２．９に調整した後、分画分子量４万材質ポリアクリロニトリルの限外ろ過膜ＤＵＹＬ−０００［ダイセル化学工業社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 19
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 2.9, the circulation linear velocity 0.5 m at 5 m 2 of ultrafiltration membrane DUYL-000 [manufactured by Daicel Chemical Industries, Ltd.] having a molecular weight cut off of 40,000. / S, volume ratio concentration double concentration, and equal-pure pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 2.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２０
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［冨田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．９に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して９４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 20
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Iwata Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Company Limited] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in Step A to pH 3.9, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-fold pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 2.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 94 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２１
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 21
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２２
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［冨田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ１６×８４０ｍｍＣＥＬＡＦＬＯ［日本ミリポア・リミテッド社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 22
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Iwata Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Company Limited] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 70 l per 1 m 2 is applied to a ceramic membrane filter φ16 × 840 mm CELAFLO (manufactured by Nihon Millipore Limited) made of alumina with a filtration accuracy of 1.0 μm. The liquid was passed at a flow rate of / hr.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２３
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ１０×１０００ｍｍ マルチポアロンＴＣ−２［三井研削砥石社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 23
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m is applied to a ceramic membrane filter φ10 × 1000 mm made of alumina having a filtration accuracy of 2.0 μm, Multiporeron TC-2 [manufactured by Mitsui Grinding Stone Co., Ltd.] The liquid was passed at a flow rate of 70 l / hr per two .
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２４
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．５μｍのアルミナからなるセラミック膜フィルターφ３１×７５０ｍｍ ＭＥＭＢＲＡＬＯＸ チューブタイプ メディア［東芝セラミックス社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 24
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, an effective filtration surface area of 1 m 2 is applied to a ceramic membrane filter φ31 × 750 mm MEMBRALOX tube type media [manufactured by Toshiba Ceramics Co., Ltd.] made of alumina with a filtration accuracy of 1.5 μm. The liquid was passed at a flow rate of 70 l / hr.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a flow rate of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２５
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ＰＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基をシリカゲルに化学的に結合させた逆相クロマトグラフィー用充填剤ＹＭＣ・ＧＥＬＯＤＳ−ＡＱ−１２０−Ｓ５０［ＹＭＣ社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４０ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 25
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, PH 4.0) cultured using Streptococcus equii FM-100 (Microtechnological Laboratories 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] is effective The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of the filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is packed with 1.0 liter of YMC · GELODS-AQ-120-S50 [manufactured by YMC Co., Ltd.], which has a chemical bond of octadecyl group to silica gel. Then, ethanol, water, and sodium chloride solution were equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed with 8 l of ethanol three times, and vacuum dried at 40 ° C. to obtain 140 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２６
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤Ｏｃｔａｄｅｃｙｌ−４ＰＷ［東ソー社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒナルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 26
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography filler Octadecyl-4PW [manufactured by Tosoh Corporation] in which an octadecyl group is chemically bonded to a synthetic resin, and water, sodium chloride The solution was equilibrated in order, and 70 l of the solution obtained in Step C was passed through the column at a flow rate of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed with 8 l of ethanol three times, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２７
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ ７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍ樹脂製カラムに、γ−メチル Ｌ−グルタメートをアミノ化した充填剤パイロトール［日本濾水機工業社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 27
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD 700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%), Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon, and using a stirrer, 50 rpm The suspension was stirred for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm was charged with 1.0 l of a filler Pyrotol (manufactured by Nippon Seiki Kogyo Co., Ltd.) aminated with γ-methyl L-glutamate, and equilibrated in the order of water and sodium chloride solution. 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２８
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、セルロースを基材とした充填剤パイロセップ−Ｃ［ダイセル化学工業社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンプランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 28
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 liter of cellulose-based filler Pyrosep-C [manufactured by Daicel Chemical Industries, Ltd.], equilibrated in the order of water and sodium chloride solution, 70 l of the solution obtained in Step C was passed at a flow rate of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in the step D was passed through a positivedyne menplan filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例２９
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、キトサンを基材とした充填剤クリムーバＩＩ［栗田工業社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 29
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 liter of chitosan-based filler Crimover II [manufactured by Kurita Kogyo Co., Ltd.], equilibrated in the order of water and sodium chloride solution, and step C is added to the column. 70 l of the solution obtained in the above was passed at a flow rate of a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３０
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．２μｍのプラスのゼータ電位を有するポジダイン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 30
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in the step D was passed through a positivedyne membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.2 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３１
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度１μｍのプラスのゼータ電位を有するプロファイルプラス・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 31
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a profile plus membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 1 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３２
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．２μｍのプラスのゼータ電位を有するウルチポアＧＦプラス・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１３９ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 32
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed at a flow rate of 220 l / hr per m 2 of effective filtration surface area through a Ultipore GF plus membrane filter (manufactured by Nippon Pole) having a positive zeta potential with a filtration accuracy of 0.2 μm. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 139 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３３
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍの疎水性膜デュラポア・メンブランフィルター［日本ミリポア社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 33
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a hydrophobic membrane Durapore membrane filter (manufactured by Nihon Millipore) with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３４
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン醸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｒｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．２μｍの疎水性膜エンフロン・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４１ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 34
[Step A]
45 l of fermentation broth (hyaluronic sodium concentration 3.5 g / l, pH 4.0) cultured with Streptococcus equii FM-100 (MICROKEN KENYO 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a hydrophobic membrane Enflon membrane filter (manufactured by Nippon Pole Co., Ltd.) having a filtration accuracy of 0.2 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 141 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３５
［Ａ工程］
ストレプトコッカス・工クイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［冨田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．２μｍのポリアミド膜ウルチポアＮ６６・メンブランフィルター［日本ポール社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 35
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus / Kuiku FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank and inorganic adsorbent Tomix AD-700NS [made by Iwata Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%), and as activated carbon, Shirakaba P [made by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) are added, and a stirrer is added. Using suspension at 50 rpm for 0.5 hour, the suspension was stirred. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a polyamide membrane Ultipore N66 membrane filter (manufactured by Nippon Pole Co., Ltd.) having a filtration accuracy of 0.2 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３６
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．２μｍのポリアミド膜ゼータポア・メンブランフィルター［キュノ社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４３ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 36
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a polyamide membrane zetapore membrane filter (manufactured by Cuno) with a filtration accuracy of 0.2 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 143 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３７
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［冨田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．２μｍのポリスルフォン膜スーポアフロー・メンブランフィルター［ゲルマンサイエンス社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４２ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 37
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Iwata Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Company Limited] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a polysulfone membrane super flow membrane filter (manufactured by Gelman Science) with a filtration accuracy of 0.2 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 142 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３８
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度１．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤ＯＤＰ４００［昭和電工社製］を１．０ｌ充填し、エタノール、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍのポリスルフォン膜スーポアフロー・メンブランフィルター［ゲルマンサイエンス社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４２ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 38
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 1.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography packing ODP400 [manufactured by Showa Denko KK] in which an octadecyl group is chemically bonded to a synthetic resin, followed by ethanol, water, and chloride. The sodium solution was equilibrated in this order, and 70 l of the solution obtained in step C was passed through the column at a flow rate of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a polysulfone membrane super flow membrane filter (manufactured by Gelman Science) with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 142 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例３９
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり８５ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤Ｏｃｔａｄｅｃｙｌ−４ＰＷ［東ソー社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．８５ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍの親水性膜デュラポア・メンブランフィルター［日本ミリポア社製］に有効ろ過表面積１ｍ２あたり２６０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４２ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 39
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 2.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 85 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography filler Octadecyl-4PW [manufactured by Tosoh Corporation] in which an octadecyl group is chemically bonded to a synthetic resin, and water, sodium chloride The solution was equilibrated in the order, and 70 l of the solution obtained in Step C was passed through the column at a linear flow rate of 0.85 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a hydrophilic membrane Durapore membrane filter (manufactured by Nippon Millipore) with a filtration accuracy of 0.45 μm at a flow rate of 260 l / hr per m 2 of effective filtration surface area. The obtained solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 142 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例４０
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり４５ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤Ｏｃｔａｄｅｃｙｌ−４ＰＷ［東ソー社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．４５ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍの親水性膜デュラポア・メンブランフィルター［日本ミリポア社製］に有効ろ過表面積１ｍ２あたり１４５ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 40
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 2.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 45 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography filler Octadecyl-4PW [manufactured by Tosoh Corporation] in which an octadecyl group is chemically bonded to a synthetic resin, and water, sodium chloride The solution was equilibrated in the order, and 70 l of the solution obtained in step C was passed through the column at a linear flow rate of 0.45 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a hydrophilic membrane Durapore membrane filter (manufactured by Nippon Millipore) with a filtration accuracy of 0.45 μm at a flow rate of 145 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例４１
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製」０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤Ｏｃｔａｄｅｃｙｌ−４ＰＷ［東ソー社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．７ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍの親水性膜デュラポア・メンブランフィルター［日本ミリポア社製］に有効ろ過表面積１ｍ２あたり２２０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 41
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [produced by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [produced by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 2.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 70 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography filler Octadecyl-4PW [manufactured by Tosoh Corporation] in which an octadecyl group is chemically bonded to a synthetic resin, and water, sodium chloride The solution was equilibrated in order, and 70 l of the solution obtained in Step C was passed through the column at a flow rate of 0.7 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a hydrophilic membrane Durapore membrane filter (manufactured by Nippon Millipore) with a filtration accuracy of 0.45 μm at a flow rate of 220 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
実施例４２
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｃ工程］
Ａ工程で得られた溶液４５ｌを濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり８５ｌ／ｈｒの流量で通液した。
［Ｂ工程］
Ｃ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｄ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤Ｏｃｔａｄｅｃｙｌ−４ｐｗ［東ソー社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＢ工程で得られた溶液７０ｌをＳ線速０．８５ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍの親水性膜デュラポア・メンブランフィルター［日本ミリポア社製］に有効ろ過表面積１ｍ２あたり２６０ｌ／ｈｒの流量で通液した。得られた溶液をエタノール２１０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して１４４ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Example 42
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step C]
45 l of the solution obtained in the step A is applied to a ceramic membrane filter φ30 × 1000 mm, φ4 × 19 hole, NGK monolith type [manufactured by NGK Corporation] made of alumina with a filtration accuracy of 2.0 μm, and an effective filtration surface area of 85 l / hr per 1 m 2. The liquid was passed at a flow rate.
[Step B]
After adjusting 45 l of the solution obtained in Step C to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of an ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-fold pure water dilution were repeatedly treated with 11 dialyzes.
[D process]
Packing agent for reverse phase chromatography in which 2.1 kg of sodium chloride is dissolved in 70 l of the solution obtained in step B, and then a octadecyl group is chemically bonded to a synthetic resin in a resin column having an inner diameter of 25 cm and a height of 2 cm. Octadecyl-4pw [manufactured by Tosoh Corporation] is charged in an amount of 1.0 l, equilibrated in the order of water and sodium chloride solution, and 70 l of the solution obtained in step B is applied to the column at an S linear velocity of 0.85 m 3 / m 2 · hr. The liquid was passed at a flow rate.
[E process]
70 l of the solution obtained in step D was passed through a hydrophilic membrane Durapore membrane filter (manufactured by Nippon Millipore) with a filtration accuracy of 0.45 μm at a flow rate of 260 l / hr per m 2 of effective filtration surface area. The resulting solution was precipitated with 210 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. to obtain 144 g of sodium hyaluronate. The analysis results are shown in Table 1.
比較例１
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ３．５）を２００ｌのポリエチレン製タンクに採取する。前記処理液を濾紙（アドバンテック東洋社製１０３４−２）で加圧による濾過処理を行った。得られた溶液に塩化ナトリウム１．３４ｋｇを溶解、ｐＨ７に調整後、エタノール１３５ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥した。分析結果を表１に示す。
Comparative Example 1
45 l of a fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 3.5) cultured using Streptococcus equii FM-100 (Microtechnological Laboratory 9027) is collected in a 200 l polyethylene tank. The treatment liquid was subjected to filtration treatment by pressurization with filter paper (1034-2 manufactured by Advantech Toyo Co., Ltd.). 1.34 kg of sodium chloride was dissolved in the resulting solution, adjusted to pH 7, precipitated with 135 l of ethanol, washed 3 times with 8 l of ethanol, and vacuum dried at 40 ° C. The analysis results are shown in Table 1.
比較例２
［Ａ工程］
ストレプトコッカス・エクイＦＭ−１００（微工研菌寄９０２７）を用いて培養した発酵液４５ｌ（ヒアルロン酸ナトリウム濃度３．５ｇ／ｌ、ｐＨ４．０）を１００ｌのポリエチレン製タンクに採取し無機吸着剤としてトミックスＡＤ−７００ＮＳ［富田製薬社製］０．４５０ｋｇ（１．０重量％）、活性炭として白鷺Ｐ［武田薬品工業社製］３．１５０ｋｇ（７．０重量％）を添加し、撹拌機を用い５０ｒｐｍで０．５時間懸濁撹拌処理した。前記処理液を濾紙１０３４−２［アドバンテック東洋社製］で加圧による濾過処理を行った。
［Ｂ工程］
Ａ工程で得られた溶液４５ｌをｐＨ３．５に調整した後、分画分子量１．３万材質ポリスルフォンの限外ろ過膜ＭＵ−６３０３−ＨＧ［クラレ社製］５ｍ２にて循環線速度０．５ｍ／ｓ、容積比２倍濃縮、等倍純水希釈を繰り返し透析回数１１回で処理した。
［Ｃ工程］
Ｂ工程で得られた溶液７０ｌに塩化ナトリウム２．１ｋｇを溶解した後、濾過精度２．０μｍのアルミナからなるセラミック膜フィルターφ３０×１０００ｍｍ、φ４×１９穴、ＮＧＫ モノリス型［日本ガイシ社製］に有効ろ過表面積１ｍ２あたり２５ｌ／ｈｒの流量で通液した。
［Ｄ工程］
内径２５ｃｍ、高さ２ｃｍの樹脂製カラムに、オクタデシル基を合成樹脂に化学的に結合させた逆相クロマトグラフィー用充填剤Ｏｃｔａｄｅｃｙｌ−４ＰＷ［東ソー社製］を１．０ｌ充填し、水、塩化ナトリウム溶液の順に平衡化し、上記カラムにＣ工程で得られた溶液７０ｌを線速０．２５ｍ３／ｍ２・ｈｒの流量で通液した。
［Ｅ工程］
Ｄ工程で得られた溶液７０ｌを濾過精度０．４５μｍの親水性膜デュラポア・メンブランフィルター［日本ミリポア社製］に有効ろ過表面積１ｍ２あたり７０ｌ／ｈｒの流量で通液した。流量を速くすることで膜表面にゲル等が形成されて膜に閉塞を生じるようになったので、Ｅ工程で得られた溶液は６０ｌであった。得られた溶液をエタノール１８０ｌで析出し、エタノール８ｌで洗浄を３回行い、４０℃で真空乾燥して７９ｇのヒアルロン酸ナトリウムが得られた。分析結果を表１に示す。
Comparative Example 2
[Step A]
45 l of fermentation broth (sodium hyaluronate concentration 3.5 g / l, pH 4.0) cultivated using Streptococcus equii FM-100 (Microtechnological Bacteria 9027) was collected in a 100 l polyethylene tank as an inorganic adsorbent. Tomix AD-700NS [manufactured by Tomita Pharmaceutical Co., Ltd.] 0.450 kg (1.0 wt%) and Shirakaba P [manufactured by Takeda Pharmaceutical Co., Ltd.] 3.150 kg (7.0 wt%) as activated carbon were added, and a stirrer was used. The suspension was stirred and stirred at 50 rpm for 0.5 hour. The treatment liquid was subjected to filtration treatment by pressurization with filter paper 1034-2 [manufactured by Advantech Toyo Co., Ltd.].
[Step B]
After adjusting 45 liters of the solution obtained in step A to pH 3.5, the circulation linear velocity was 0 at 5 m 2 of ultrafiltration membrane MU-6303-HG (manufactured by Kuraray Co., Ltd.) having a molecular weight cut off of 13,000. 0.5 m / s, volume ratio double concentration, and equal-pure water dilution were repeatedly treated with 11 dialyzes.
[Step C]
After dissolving 2.1 kg of sodium chloride in 70 l of the solution obtained in step B, the ceramic membrane filter made of alumina with a filtration accuracy of 2.0 μm φ30 × 1000 mm, φ4 × 19 holes, NGK monolith type [manufactured by NGK Corporation] The liquid was passed at a flow rate of 25 l / hr per 1 m 2 of effective filtration surface area.
[D process]
A resin column having an inner diameter of 25 cm and a height of 2 cm is filled with 1.0 l of a reverse phase chromatography filler Octadecyl-4PW [manufactured by Tosoh Corporation] in which an octadecyl group is chemically bonded to a synthetic resin, and water, sodium chloride The solution was equilibrated in the order, and 70 l of the solution obtained in step C was passed through the column at a linear velocity of 0.25 m 3 / m 2 · hr.
[E process]
70 l of the solution obtained in step D was passed through a hydrophilic membrane Durapore membrane filter (manufactured by Nihon Millipore) with a filtration accuracy of 0.45 μm at a flow rate of 70 l / hr per m 2 of effective filtration surface area. Since the gel was formed on the film surface by increasing the flow rate and the film was clogged, the solution obtained in the E step was 60 l. The obtained solution was precipitated with 180 l of ethanol, washed with 8 l of ethanol three times, and vacuum-dried at 40 ° C. to obtain 79 g of sodium hyaluronate. The analysis results are shown in Table 1.
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
＊ Ｃａ、Ｆｅ、Ｍｇ、Ｃｒ、Ｓｉ、Ａｌ、Ｃｕ、Ｐｂの合計組成
N.D. 検出せず
* Total composition of Ca, Fe, Mg, Cr, Si, Al, Cu, Pb
ND not detected
測定方法
（１）タンパク質：精製ヒアルロン酸を０．１N水酸化ナトリウムに溶かし、ローリー法でタンパク質含量を測定した。
（２）乳酸：ヒアルロン酸ナトリウムを０．１％の濃度に溶解し、Ｌ−ＬＤＨ法にて行った。
（３）核酸：０．１％ヒアルロン酸ナトリウムの２６０ｎｍにおける吸光度を測定した。
（４）アミノ酸：ヒアルロン酸ナトリウムを０．１％濃度に溶解し、ＬＣ−ＯＰＡ法にて行った。
（５）エンドトキシン：生化学工業社製トキシカラーシステムにより比色分析した。
（６）重金属：ヒアルロン酸ナトリウムを０．１ｇ白金るつぼにとり、局方第二法にて行った。
（７）金属：ヒアルロン酸ナトリウムを０．０５％の濃度になるように８Ｎ硝酸に溶解し、ＩＣＰ発光分光分析を行った。
（８）異物：ヒアルロン酸ナトリウムを０．１％となるように、ペプトン緩衝液に溶解し、日本薬局方、一般試験法の無菌試験・メンブレンフィルター法で行った。
（９）極限粘度：ヒアルロン酸ナトリウムを０．０２％となるように、０．２Ｍ塩化ナトリウムに溶解し、３０℃における極限粘度を測定した。
Measurement method (1) Protein: Purified hyaluronic acid was dissolved in 0.1N sodium hydroxide, and the protein content was measured by the Raleigh method.
(2) Lactic acid: Sodium hyaluronate was dissolved to a concentration of 0.1%, and the L-LDH method was used.
(3) Nucleic acid: The absorbance at 260 nm of 0.1% sodium hyaluronate was measured.
(4) Amino acid: Sodium hyaluronate was dissolved to a concentration of 0.1%, and the LC-OPA method was used.
(5) Endotoxin: Colorimetric analysis was performed using a Toxicolor system manufactured by Seikagaku Corporation.
(6) Heavy metal: 0.1 g of sodium hyaluronate was placed in a platinum crucible and subjected to the second method of the Japanese Pharmacopoeia.
(7) Metal: Sodium hyaluronate was dissolved in 8N nitric acid to a concentration of 0.05%, and ICP emission spectroscopic analysis was performed.
(8) Foreign matter: Sodium hyaluronate was dissolved in peptone buffer so that the concentration was 0.1%, and the sterility test and membrane filter method of the Japanese Pharmacopoeia and general test methods were performed.
(9) Intrinsic viscosity: Sodium hyaluronate was dissolved in 0.2 M sodium chloride so as to be 0.02%, and the intrinsic viscosity at 30 ° C. was measured.
Claims (47)
Ａ工程：無機吸着剤、又は無機吸着剤と活性炭にヒアルロン酸及び／又はその塩の含有液を接触させる工程。
Ｂ工程：ヒアルロン酸及び／又はその塩の含有液を酸性側のｐＨ範囲内に調整した後、限外ろ過膜にて透析処理する工程。
Ｃ工程：ヒアルロン酸及び／又はその塩の含有液をセラミックス膜に接触させる工程。
Ｄ工程：ヒアルロン酸及び／又はその塩の含有液を天然物を基材としたクロマトグラフィー用充填剤又は、担体に鎖長が炭素数８以上の置換又は未置換の炭化水素基を結合させた逆相クロマトグラフィー用充填剤に接触させる工程。
Ｅ工程：ヒアルロン酸及び／又はその塩の含有液を精密ろ過膜に接触させる工程。 A method for purifying hyaluronic acid and / or a salt thereof, comprising the following five steps of A, B, C, D and E.
Step A: A step of bringing an inorganic adsorbent or a liquid containing hyaluronic acid and / or a salt thereof into contact with the inorganic adsorbent and activated carbon.
Step B: A step of adjusting the solution containing hyaluronic acid and / or a salt thereof within the pH range on the acidic side, followed by dialysis treatment with an ultrafiltration membrane.
Step C: A step of bringing a liquid containing hyaluronic acid and / or a salt thereof into contact with the ceramic film.
Step D: A substituted or unsubstituted hydrocarbon group having a chain length of 8 or more is bonded to a chromatographic packing material or carrier based on hyaluronic acid and / or a salt thereof, or a carrier. Contacting with a packing for reverse phase chromatography.
Step E: A step of bringing a hyaluronic acid and / or salt-containing liquid into contact with a microfiltration membrane.
Ａ工程： 請求項１４記載の無機吸着剤と水蒸気賦活活性炭、ウェットな活性炭、粉末活性炭、粒状活性炭、薬品賦活活性炭、石炭由来活性炭、木材由来活性炭、ドライな活性炭から選ばれた少なくとも一種の混合物にヒアルロン酸及び／又はその塩の含有液を接触させる工程。
Ｂ工程： Ａ工程で得られた溶液を酢酸セルロース、ポリアクリロニトリル、ポリスルフォン、ポリエーテルスルフォン、ハロゲン化ポリオレフィン、ポリアミド、ポリフッ化ビニリデンから選ばれた材質よりなる限外ろ過膜にて酸性側のｐＨ範囲内に調整した後、透析処理する工程。
Ｃ工程： Ｂ工程で得られた溶液をＡｌ２Ｏ３、ＺｒＯ３、ＳｉＯ２、ＴｉＯ２、Ｓｉ３Ｎ４、ＳｉＣ、ＢＮ、ＭｇＯ、及びＺｎＯ等のある適当な配合比の材質よりなるセラミック膜に接触させる工程。
Ｄ工程： Ｃ工程で得られた溶液をセルロース、ヒスチジン、メチルグルタメート、キチンキトサン等の天然物を基材とするクロマトグラフィー用充填剤又は、炭素数８以上の置換又は未置換の炭化水素基特にオクチル基およびこれらの末端を改変した官能基、オクタデシル基およびこれらの末端を改変した官能基から選ばれた一種の官能基を有する逆相クロマトグラフィー用充填剤に接触させる工程。
Ｅ工程： Ｄ工程で得られた溶液をポリスルフォン等からなる親水性膜、６−ナイロン、６，６−ナイロン、７−ナイロン、１１−ナイロン、１２−ナイロン、６，１１−ナイロン、６，１２−ナイロン等からなるポリアミド膜、ポリビニリデンジフルオライド、ポリテトラエチレン、ポリエチレン、ポリプロピレン等からなる疎水性膜、プラスのゼータ電位を有する膜から選ばれたものに接触させる工程。 A method for purifying hyaluronic acid and / or a salt thereof, which is processed in the order of the following five steps of A, B, C, D and E.
Step A: To at least one mixture selected from the inorganic adsorbent according to claim 14, water vapor activated activated carbon, wet activated carbon, powdered activated carbon, granular activated carbon, chemical activated activated carbon, coal-derived activated carbon, wood-derived activated carbon, and dry activated carbon. A step of contacting a liquid containing hyaluronic acid and / or a salt thereof.
Step B: The solution obtained in Step A is subjected to acidic pH in an ultrafiltration membrane made of a material selected from cellulose acetate, polyacrylonitrile, polysulfone, polyether sulfone, halogenated polyolefin, polyamide, and polyvinylidene fluoride. A process of dialysis after adjustment within the range.
Step C: A ceramic made of a material having an appropriate mixing ratio such as Al 2 O 3 , ZrO 3 , SiO 2 , TiO 2 , Si 3 N 4 , SiC, BN, MgO, and ZnO. Contacting the membrane.
Step D: The solution obtained in Step C is a chromatographic filler based on natural products such as cellulose, histidine, methyl glutamate, and chitin chitosan, or a substituted or unsubstituted hydrocarbon group having 8 or more carbon atoms, particularly Contacting with a packing material for reverse phase chromatography having one kind of functional group selected from an octyl group and functional groups modified at these ends, octadecyl group and functional groups modified at these ends.
Step E: The solution obtained in Step D is a hydrophilic film made of polysulfone or the like, 6-nylon, 6,6-nylon, 7-nylon, 11-nylon, 12-nylon, 6,11-nylon, 6, A step of contacting a polyamide film made of 12-nylon or the like, a hydrophobic film made of polyvinylidene difluoride, polytetraethylene, polyethylene, polypropylene, or the like, or a film having a positive zeta potential.
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| CN103319627A (en) * | 2013-07-10 | 2013-09-25 | 南宁中诺生物工程有限责任公司 | Method for grading molecule weight of sodium hyaluronate |
| CN104804105A (en) * | 2015-04-08 | 2015-07-29 | 成都郫县新星成明食品研究所 | Method for preparing carrageenan according to ceramic membrane filtration method |
| CN106397630A (en) * | 2016-08-31 | 2017-02-15 | 新疆阜丰生物科技有限公司 | Method for extracting sodium hyaluronate based on membrane separation technology |
| CN108383196A (en) * | 2018-04-02 | 2018-08-10 | 合肥卓畅农业科技有限公司 | Antibacterial decontamination environment-friendly high-efficiency water purification agent of one kind and preparation method thereof |
| CN110577608A (en) * | 2019-07-04 | 2019-12-17 | 东莞东阳光药物研发有限公司 | Method for separating and purifying hyaluronic acid |
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