JP3560066B2 - Method for fractionating plasma protein - Google Patents
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Description
【０００１】
【産業上の利用分野】
本発明は血漿蛋白含有物、特に血漿から血漿蛋白を効率よく各種血漿蛋白成分に分画する方法に関する。
【０００２】
【従来の技術】
血漿蛋白は血漿に含まれる蛋白の総称であり、１００種類以上の蛋白成分からなる。血漿蛋白の主な成分はアルブミン、グロブリン、各種血液凝固因子、フィブリノーゲン、フィブロネクチン、プラスミノーゲン、プロトロンビン等である。血漿からこれらの血漿蛋白成分に分画する方法としては、コーンの低温エタノール分画法、硫安分画法、ポリエチレングリコール分画法等が知られている。
【０００３】
ところで、低温エタノール分画法において、生成された上清画分および沈殿画分を分離する方法としては遠心分離法、濾過分離法等が知られている。このような低温エタノール分画法としては、例えば、Ｐｒｏｃｅｓｓ Ｂｉｏｃｈｅｍ．，７，２０（１９７２）、Ｖｏｘ Ｓａｎｇ，３１，２８９−２９５（１９７６）等に記載がある。
【０００４】
遠心分離法は遠心機１台当たりの処理能力に限界がある点や、高速回転に伴う発熱および騒音、あるいは高速回転体による危険性といった問題点のあることが指摘されている。そこで、このような問題点を回避することを目的として、濾過処理法による実用化が展開されつつある。
【０００５】
【発明が解決しようとする課題】
本発明は従来の遠心分離法に比較して、血漿蛋白含有物、特に血漿から血漿蛋白を安全かつ効率よく分画することができる方法を提供することを目的とするものである。
【０００６】
【課題を解決するための手段】
本発明者らは上記の事情を考慮して各種検討を行った結果、低温エタノール分画処理により調製された上清画分および沈殿画分を、特定の条件下において濾過処理することにより効率よく分離回収することに成功し、本発明を完成した。本発明は、以下の血漿蛋白の分画方法であり、これらにより上記目的を達成できる。
１） 血漿蛋白含有物をエタノール濃度５〜１０％、ｐＨ６．８〜７．４の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積５〜１５ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下の条件下に濾過処理を行うことにより第Ｉ沈殿画分および第Ｉ上清画分を分離回収することを特徴とする血漿蛋白の分画方法。
２） 第Ｉ上清画分をエタノール濃度１８〜３０％、ｐＨ４．５〜８の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積２０〜３０ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下の条件下に濾過処理を行うことにより第ＩＩ＋ＩＩＩ沈殿画分および第ＩＩ＋ＩＩＩ上清画分を分離回収することを特徴とする血漿蛋白の分画方法。
３） 第ＩＩ＋ＩＩＩ上清画分をエタノール濃度３５〜４５％、ｐＨ５〜７の条件下での低温分画処理後に孔径０．４〜２μｍ、濾過面積１０〜２０ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下、濾過助剤を１〜１００ｇ（液量１Ｌ当たり）添加の条件下に濾過処理を行うことにより第ＩＶ沈殿画分および第ＩＶ上清画分を分離回収することを特徴とする血漿蛋白の分画方法。
４） 第ＩＶ上清画分をエタノール濃度３５〜４５％、ｐＨ４〜５．５の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積２０〜３０ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下に濾過処理を行うことにより第Ｖ沈殿画分および第Ｖ上清画分を分離回収することを特徴とする血漿蛋白の分画方法。
５） 孔径０．６〜２μｍの条件下に濾過処理を行う上記１）記載の血漿蛋白の分画方法。
６） 孔径０．６〜２μｍの条件下に濾過処理を行う上記２）記載の血漿蛋白の分画方法。
７） 孔径０．４〜１μｍの条件下に濾過処理を行う上記３）記載の血漿蛋白の分画方 法。
８） 孔径０．６〜２μｍの条件下に濾過処理を行う上記４）記載の血漿蛋白の分画方法。
９） 血漿蛋白含有物をエタノール濃度５〜１０％、ｐＨ６．８〜７．４の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積５〜１５ｍ 2 （液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ 2 以下の条件下に濾過処理を行うことにより第Ｉ沈殿画分および第Ｉ上清画分を分離回収し、
第Ｉ上清画分をエタノール濃度１８〜３０％、ｐＨ４．５〜８の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積２０〜３０ｍ 2 （液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ 2 以下の条件下に濾過処理を行うことにより第ＩＩ＋ＩＩＩ沈殿画分および第ＩＩ＋ＩＩＩ上清画分を分離回収し、
第ＩＩ＋ＩＩＩ上清画分をエタノール濃度３５〜４５％、ｐＨ５〜７の条件下での低温分画処理後に孔径０．４〜２μｍ、濾過面積１０〜２０ｍ 2 （液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ 2 以下、濾過助剤を１〜１００ｇ（液量１Ｌ当たり）添加の条件下に濾過処理を行うことにより第ＩＶ沈殿画分および第ＩＶ上清画分を分離回収し、
第ＩＶ上清画分をエタノール濃度３５〜４５％、ｐＨ４〜５．５の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積２０〜３０ｍ 2 （液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ 2 以下に濾過処理を行うことにより第Ｖ沈殿画分および第Ｖ上清画分を分離回収することを特徴とする血漿蛋白の分画方法。
【０００７】
本発明は、血漿蛋白含有物もしくはその画分を低温エタノール分画処理し、次いで特定の条件下において濾過処理することにより効率よく低温エタノール分画処理により生成した上清画分と沈殿画分をそれぞれ分離回収するものであり、本発明の特徴は該濾過処理の最適条件を見出したことにある。本発明における濾過処理条件は、前記した通りである。そして、本発明は該条件を満足するように従来公知の濾過手段（濾過材、および濾過装置）を適宜選択し得る。
【０００８】
濾過処理条件における孔径とは、濾過材の平均孔径を意味し、濾過面積とは濾過すべき液１０００〜５０００Ｌ（リットル）を柱体形状で保持している濾過材表面の面積（即ち、柱体の横断面積または底面）を指す。また、必要により濾過助剤を適宜使用することができる。この場合、濾過助剤は主として沈殿物を多孔質化し、濾過抵抗を著しく減少させる作用を有するものであり、濾過材の濾過機能を促進する機能を有する。濾過助剤は低温エタノール分画処理した液に濾過前もしくは濾過と同時に添加される。そして、本発明では、該濾過すべき液を圧力１Ｋｇ／ｃｍ2以下で濾過する。
【０００９】
〔１〕出発原料
本発明の出発原料として使用される血漿蛋白含有物は、血漿蛋白を含有するものであれば、特に限定されないが、通常、例えば、血液、血漿、血清、これらに由来する画分等が挙げられる。具体的には血漿からクリオプレシピテートを除去したもの、血漿からプロトロンビン複合体を除去したもの等が例示される。
【００１０】
本発明１）においては、濾過処理により第Ｉ沈殿画分と第Ｉ上清画分が得られる。これは従来法によるコーンの第Ｉ沈殿画分およびその上清画分に相当することが、後述の実験例からも明らかであるが、従来の画分成分と厳密に一致しなければならないものではない。従って、本発明２）に使用される第Ｉ上清画分は、本発明１）からのものに限定されずこれと実質的に変わらない従来法によるコーンの第Ｉ上清画分であってもかまわない。
【００１１】
同様に、本発明３）に使用される第ＩＩ＋ＩＩＩ上清画分、または本発明４）で使用される第ＩＶ上清画分も従来法のコーンの各画分に相当するので、本発明２）の第Ｉ上清画分と同様な取扱いができる。
〔２〕本発明の濾過処理（フィルタープレス使用）
（１）第Ｉ沈殿画分と同上清画分の分離
出発原料をエタノール濃度５〜１０％、好ましくは７〜９％で処理して上清画分と沈殿画分とに分離する。沈殿画分よりフィブリノゲン等を回収することができる。
【００１２】
１．エタノール処理条件
ｐＨ６．８〜７．４、温度５〜−３℃（特に−２〜−３℃）、３０分間〜１５時間特に１〜５時間）程度が例示される。なお、エタノール処理条件は公知のコーンの低温エタノール分画法に準じている。以下、（２）〜（４）も同様である。
【００１３】
２．濾過処理条件
ａ．孔径０．６〜４μｍ、濾過面積５〜１５ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下、濾過時間１０時間以内の条件下に濾過処理を行うことが例示される。このうち、最も好ましいのは０．６〜２μｍの孔径幅を有する濾過材（例、ゼータプラス３０ＬＡ）であるが、０．９〜４μｍの孔径幅を有する濾過材（例、ゼータプラス１０ＬＡ）も同程度に好ましい。好ましい圧力は１ｋｇ／ｃｍ2である。なお、濾過時間は濾過面積、濾過液量に応じて適宜変更することができる。また、濾過助剤を１〜１００ｇ（液量１Ｌ当たり）程度添加することが好ましい。濾過助剤としてはケイソウ土等が例示される。
【００１４】
ｂ．フィルタープレス
フィルタープレスは濾過装置および濾過材から構成される。濾過装置は加圧濾過式、圧搾式、ガスブロー式、電気浸透式、それらの組合せ等が知られている。また、濾過装置は、市販品を使用できる。具体的には加圧濾過とガスブローを組合わた濾過装置（ＳＥＩＴＺ社製、商品名ＯＲＩＯＮ Ｃ４０）等が例示される。
【００１５】
濾過材（濾布を含む概念である）は繊維（例、セルロース等）、樹脂、無機濾過助剤（ケイソウ土、パーライト等）等が知られている。この濾過材は市販品を使用できる。具体的には高度精製セルロース繊維と酸処理ケイソウ土を組合せた濾過材（キュノ社製ゼータプラスＬＡシリーズ）等が例示される。尚、以下の（２）〜（４）においても上記と同様のフィルタープレスを使用することができる。
【００１６】
（２）第ＩＩ＋ＩＩＩ沈殿画分と同上清画分の分離
第Ｉ上清画分をエタノール濃度１８〜３０％、好ましくは２０〜２５％で処理して上清画分と沈殿画分とに分離する。沈殿画分より免疫グロブリン等を回収することができる。
１．エタノール処理条件
ｐＨ４．５〜８（特にｐＨ５．２〜７）、温度０〜−７℃（特に−５〜−６℃）、３０分間〜１５時間（特に１〜５時間）程度が例示される。
【００１７】
２．濾過処理条件
孔径０．６〜４μｍ、濾過面積２０〜３０ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下、濾過時間１０時間以内の条件下に濾過処理を行うことが例示される。このうち、最も好ましいのは０．６〜２μｍの孔径幅を有する濾過材（例、ゼータプラス３０ＬＡ）であるが、０．９〜４μｍの孔径幅を有する濾過材（例、ゼータプラス１０ＬＡ）も同程度に好ましい。好ましい圧力は１ｋｇ／ｃｍ2である。また、濾過助剤を使用することもできる。
【００１８】
（３）第ＩＶ沈殿画分と同上清画分の分離
第ＩＩ＋ＩＩＩ上清画分をエタノール濃度３５〜４５％で処理して上清画分と沈殿画分とに分離する。沈殿画分よりアンチトロンビン−ＩＩＩ、ハプトグロビン等を回収することができる。
１．エタノール処理条件
ｐＨ５〜７（特にｐＨ５．８〜６．３）、温度０〜−７℃（特に−５〜−６℃）、３０分間〜１５時間（特に１〜５時間）程度が例示される。
【００１９】
２．濾過処理条件
孔径０．４〜２μｍ、濾過面積２０〜３０ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下、濾過時間１０時間以内の条件下に濾過処理を行うことが例示される。このうち、最も好ましいのは０．４〜１μｍの孔径幅を有する濾過材（例、ゼータプラス５０ＬＡ）であり、次いで好ましいのは０．６〜２μｍの孔径幅を有する濾過材（例、ゼータプラス３０ＬＡ）である。好ましい圧力は１ｋｇ／ｃｍ2である。また、濾過助剤を１〜１００ｇ（液量１Ｌ当たり）添加する。濾過助剤としてはケイソウ土等が例示される。
【００２０】
（４）第Ｖ沈殿画分と同上清画分の分離
第ＩＶ上清画分をエタノール濃度３５〜４５％で処理して上清画分と沈殿画分とに分離する。沈殿画分よりアルブミン等を回収することができる。
１．エタノール処理条件
ｐＨ４〜５．５（特にｐＨ４．８〜５．２）、温度０〜−７℃（特に−５〜−６℃）、３０分間〜１５時間（特に１〜５時間）程度が例示される。
【００２１】
２．濾過処理条件
孔径０．６〜４μｍ、濾過面積２０〜３０ｍ2（液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ2以下、濾過時間１０時間以内の条件下に濾過処理を行うことが例示される。このうち、最も好ましいのは０．６〜２μｍの孔径幅を有する濾過材（例、ゼータプラス３０ＬＡ）であるが、０．９〜４μｍの孔径幅を有する濾過材（例、ゼータプラス１０ＬＡ）も同程度に好ましい。好ましい圧力は１ｋｇ／ｃｍ2である。また、濾過助剤を使用することもできる。このようにして分画された血漿蛋白は公知の手法により単離、精製することができる（特開平３−１２８３９８を参照のこと）。
【００２２】
【発明の効果】
本発明の方法によれば、血漿蛋白含有物、特に血漿から血漿蛋白を効率よく分画することができる。また、従来の遠心分離法に比較して、作業環境の改善（危険性、騒音等）、血漿分画能力の増強、勤務体制の改善（手間、勤務時間面）を図ることができる。
【００２３】
【実施例】
本発明をさらに詳細に説明するために実施例および実験例を挙げるが、本発明はこれらにより何ら限定されるものではない。
実施例１
血漿２０００Ｌからクリオペイスト、プロトロンビンおよび血液凝固第ＩＸ因子を分離した後の上清画分２０００Ｌに予め−２０℃に冷却されたエタノールを添加し、終濃度８％とした。ｐＨ７．２、温度−２℃で２時間静置して沈殿（第Ｉ画分）を生成した。ゼータプラス３０ＬＡ（孔径０．６〜２μｍ、キュノ社製）を用いて、濾過面積１２ｍ2、濾過圧力１Ｋｇ／ｃｍ2以下の条件下で濾過処理を行った。濾過助剤としてマンビル社製酸処理セライト５３５を１０ｇ（液量１Ｌ当たり）使用した。濾過装置は、ＳＥＩＺＴ社製、商品名ＯＲＩＯＮ Ｃ４０（全長２９２０ｍｍ×幅７２０ｍｍ×高さ９８０ｍｍ）を用いた。濾過処理は３時間で終了した。沈殿画分は、フィブリノゲン製剤の原料となる。上清画分は次の実施例に用いた。
【００２４】
実施例２
第Ｉ上清画分２３００Ｌに予め−２０℃に冷却されたエタノールを添加し、終濃度２１％ととした。ｐＨ６．８、温度−５℃で２時間静置して沈殿（第ＩＩ＋ＩＩＩ画分）を生成した。ゼータプラス３０ＬＡを用いて、濾過面積２４ｍ2、濾過圧力１Ｋｇ／ｃｍ2以下の条件下で濾過処理を行った。濾過処理は９時間で終了した。沈殿画分は、グロブリン製剤の原料となる。上清画分は次の実施例に用いた。
【００２５】
実施例３
第ＩＩ＋ＩＩＩ画分２７００Ｌに予め−３０℃に冷却されたエタノールを添加し、終濃度４０％とした。ｐＨ６．０、温度−５℃で２時間静置して沈殿（第ＩＶ画分）を生成した。ゼータプラス５０ＬＡ（孔径０．４〜１μｍ、キュノ社製）を用いて、濾過面積１６ｍ2、濾過圧力１Ｋｇ／ｃｍ2以下の条件下で濾過処理を行った。濾過助剤としてマンビル社製酸処理セライト５３５を２０ｇ（液量１Ｌ当たり）使用した。濾過処理は８時間で終了した。沈殿画分は、アンチトロンビン−ＩＩＩ製剤およびハプトグロビン製剤の原料となる。第ＩＶ上清画分は次の実施例に用いた。
【００２６】
実施例４
第ＩＶ上清画分４０００Ｌ（エタノール終濃度４０％）をｐＨ４．８に調整し、温度−５℃で２時間静置して沈殿（第Ｖ画分）を生成した。ゼータプラス３０ＬＡを用いて、濾過面積２４ｍ2、濾過圧力１Ｋｇ／ｃｍ2以下の条件下で濾過処理を行った。濾過処理は９時間で終了した。沈殿画分は、アルブミン製剤の原料となる。
【００２７】
実施例５
実施例３の第ＩＩ＋ＩＩＩ沈殿画分からアンチトロンビン−ＩＩＩを分離した後の画分を用いて、実施例３に準じて濾過処理を行った。調製された沈殿画分はハプトグロビン製剤の原料となる。
実験例１（処理時間）
実施例１の本発明法（濾過処理法）と同じ出発原料を同量使用して、実施例１〜４に対応した各画分を夫々、従来法（シャープレス型遠心分離装置（例、商品名シャープレスＮｏ６Ａ）を用いた遠心分離法）で上清画分と沈殿画分とを分離回収するのに必要な時間を比較検討した。尚、従来法のエタノール処理条件は、実施例と同様に行った。
【００２８】
結果を表１に示す。
【００２９】
【表１】
実験例２（収量の比較）
実験例１と同様に本発明法（濾過処理法）と従来法（遠心分離法）で各々調製された沈殿画分（ペイスト）から常法により回収できる血漿蛋白の量を比較検討してみた。量は血漿１０００Ｌ当たりの換算値で表示した。結果を表２に示す。尚、表２中、第ＩＶ画分のアンチトロンビン−ＩＩＩの「１倍」は正常人血漿１ｍｌに含まれるアンチトロンビン−ＩＩＩ量に相当する。
【００３１】
【表２】
[Industrial application fields]
The present invention relates to a method for efficiently fractionating plasma protein into various plasma protein components, particularly from plasma protein-containing substances.
[0002]
[Prior art]
Plasma protein is a general term for proteins contained in plasma and consists of 100 or more protein components. The main components of plasma proteins are albumin, globulin, various blood coagulation factors, fibrinogen, fibronectin, plasminogen, prothrombin and the like. Known methods for fractionating plasma into these plasma protein components include corn low temperature ethanol fractionation, ammonium sulfate fractionation, polyethylene glycol fractionation, and the like.
[0003]
By the way, as a method for separating the produced supernatant fraction and precipitate fraction in the low temperature ethanol fractionation method, a centrifugal separation method, a filtration separation method and the like are known. Examples of such a low-temperature ethanol fractionation method include Process Biochem. 7, 20 (1972), Vox Sang, 31, 289-295 (1976) and the like.
[0004]
It has been pointed out that the centrifugal separation method has problems such as a limit in processing capacity per centrifuge, heat generation and noise accompanying high-speed rotation, and danger due to a high-speed rotating body. Therefore, for the purpose of avoiding such problems, practically by filtration method development of Sorcerer fold.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a method capable of safely and efficiently fractionating plasma proteins from plasma protein-containing materials, particularly plasma, as compared with conventional centrifugation methods.
[0006]
[Means for Solving the Problems]
As a result of various investigations taking the above circumstances into consideration, the present inventors efficiently filtered the supernatant fraction and the precipitate fraction prepared by low-temperature ethanol fractionation treatment under specific conditions. The present invention was completed after successful separation and recovery. The present invention is the following plasma protein fractionation method, which can achieve the above object.
1) After low-temperature fractionation of the plasma protein-containing material under the conditions of an ethanol concentration of 5 to 10% and a pH of 6.8 to 7.4, a pore size of 0. The first precipitate fraction and the first supernatant fraction were obtained by filtration under conditions of 6 to 4 μm, filtration area of 5 to 15 m 2 (per 1000 to 5000 L) and pressure of 1 Kg / cm 2 or less. A method for fractionating plasma protein, comprising separating and recovering.
2) After the low temperature fractionation treatment of the first supernatant fraction under the conditions of ethanol concentration 18-30% and pH 4.5-8, the pore size was reduced to 0. By carrying out filtration under conditions of 6 to 4 μm, filtration area of 20 to 30 m 2 (per 1000 to 5000 L) and pressure of 1 Kg / cm 2 or less, the II + III precipitate fraction and the II + III supernatant fraction were obtained. A method for fractionating plasma protein, comprising separating and recovering.
3) After the II + III supernatant fraction was subjected to low-temperature fractionation under the conditions of ethanol concentration of 35 to 45% and pH of 5 to 7, the pore size was reduced to 0. Filtration is performed under the conditions of 4 to 2 μm, filtration area of 10 to 20 m 2 (per 1000 to 5000 L), pressure of 1 Kg / cm 2 or less , and addition of 1 to 100 g of filter aid (per 1 L of liquid). Thereby separating and recovering the IV precipitate fraction and the IV supernatant fraction.
4) After the low-temperature fractionation treatment of the IV supernatant fraction under the conditions of ethanol concentration of 35 to 45% and pH of 4 to 5.5, the pore size was reduced to 0. The V precipitate fraction and the V supernatant fraction are separated and recovered by performing filtration at 6 to 4 μm, a filtration area of 20 to 30 m 2 (per 1000 to 5000 L) and a pressure of 1 Kg / cm 2 or less. And a method for fractionating plasma protein.
5) The method for fractionating plasma protein according to 1) above, wherein the filtration treatment is carried out under conditions of a pore size of 0.6 to 2 μm .
6) The method for fractionating plasma protein as described in 2) above, wherein the filtration treatment is performed under the condition of a pore size of 0.6 to 2 μm .
7) fractionation how the plasma proteins above 3), wherein performing the filtering process under the conditions of pore size 0.4～1Myuemu.
8) The method for fractionating plasma protein as described in 4) above, wherein the filtration treatment is performed under the condition of a pore size of 0.6-2 μm .
9) After low-temperature fractionation treatment of plasma protein-containing material under conditions of ethanol concentration 5 to 10% and pH 6.8 to 7.4, pore size 0.6 to 4 μm, filtration area 5 to 15 m 2 (liquid amount 1000 to 5000 L) The first precipitation fraction and the first supernatant fraction are separated and recovered by filtration under a pressure of 1 kg / cm 2 or less,
The first supernatant fraction was subjected to low-temperature fractionation treatment under conditions of ethanol concentration 18-30% and pH 4.5-8, pore size 0.6-4 μm, filtration area 20-30 m 2 (per 1000 to 5000 L of liquid volume) The II + III precipitate fraction and the II + III supernatant fraction are separated and recovered by filtration under a pressure of 1 kg / cm 2 or less,
The II + III supernatant fraction was subjected to low-temperature fractionation treatment under conditions of ethanol concentration of 35 to 45% and pH of 5 to 7, pore size of 0.4 to 2 μm, filtration area of 10 to 20 m 2 (per 1000 to 5000 L), pressure The IV precipitate fraction and the IV supernatant fraction are separated and recovered by performing filtration under the condition of 1 kg / cm 2 or less and 1 to 100 g of filter aid (per 1 L of liquid volume),
The IV supernatant fraction was subjected to low-temperature fractionation treatment under conditions of ethanol concentration of 35 to 45% and pH of 4 to 5.5, a pore size of 0.6 to 4 μm, and a filtration area of 20 to 30 m 2 (per 1000 to 5000 L of liquid volume). A method for fractionating plasma protein, which comprises separating and recovering the V precipitate fraction and the V supernatant fraction by filtration at a pressure of 1 kg / cm 2 or less.
[0007]
In the present invention, a plasma protein-containing substance or a fraction thereof is subjected to a low-temperature ethanol fractionation treatment, followed by filtration under specific conditions, whereby a supernatant fraction and a precipitate fraction that are efficiently produced by the low-temperature ethanol fractionation treatment are obtained. Each of them is separated and recovered, and the feature of the present invention resides in finding the optimum conditions for the filtration treatment. Filtration conditions definitive to the onset bright is as described above. In the present invention, conventionally known filtration means (filter material and filtration device) can be appropriately selected so as to satisfy the conditions.
[0008]
The pore diameter in the filtration conditions means the average pore diameter of the filter medium, and the filtration area means the area of the filter medium surface that holds the liquid 1000 to 5000 L (liter) to be filtered in a columnar shape (that is, the column body). The cross-sectional area or bottom surface of Moreover, a filter aid can be appropriately used as necessary. In this case, the filter aid mainly has a function of making the precipitate porous and significantly reducing the filtration resistance, and has a function of promoting the filtration function of the filter medium. The filter aid is added to the liquid subjected to the low-temperature ethanol fractionation before or simultaneously with the filtration. In the present invention, the liquid to be filtered is filtered at a pressure of 1 Kg / cm 2 or less.
[0009]
[1] Starting material The plasma protein-containing material used as the starting material of the present invention is not particularly limited as long as it contains plasma protein. Usually, for example, blood, plasma, serum, and fractions derived from these Minutes. Specific examples include those obtained by removing cryoprecipitate from plasma, and those obtained by removing prothrombin complex from plasma.
[0010]
In the present invention 1) , the first precipitation fraction and the first supernatant fraction are obtained by filtration. It is clear from the experimental examples described later that this corresponds to the corn first precipitate fraction and the supernatant fraction thereof according to the conventional method, but it must exactly match the conventional fraction components. Absent. Accordingly, the first supernatant fraction used in the present invention 2) is not limited to that from the present invention 1) , but is a corn first supernatant fraction according to a conventional method which is not substantially different from this. It doesn't matter.
[0011]
Similarly, it is equal to the IV supernatant fraction also each fraction of the cone of the conventional methods used in the II + III supernatant fraction or the present invention 4) used in the present invention 3), the present invention 2 ) Can be handled in the same manner as the first supernatant fraction.
[2] Filtration treatment of the present invention (using a filter press)
(1) Separation of the first precipitate fraction and the supernatant fraction from the starting material is carried out at an ethanol concentration of 5 to 10%, preferably 7 to 9% to separate the supernatant fraction and the precipitate fraction. Fibrinogen and the like can be recovered from the precipitate fraction.
[0012]
1. Examples include ethanol treatment conditions pH 6.8 to 7.4, temperature 5 to -3 ° C (particularly -2 to -3 ° C), and 30 minutes to 15 hours, particularly 1 to 5 hours. The ethanol treatment conditions are in accordance with the known corn low temperature ethanol fractionation method. The same applies to (2) to (4) below.
[0013]
2. Filtration conditions a. Pore diameter 0. Examples of the filtration treatment include 6 to 4 μm, a filtration area of 5 to 15 m 2 (per 1000 to 5000 L), a pressure of 1 Kg / cm 2 or less, and a filtration time of 10 hours or less . Among these, the most preferable is a filter medium having a pore width of 0.6 to 2 μm (eg, zeta plus 30LA), but a filter medium having a pore size width of 0.9 to 4 μm (eg, zeta plus 10LA) is also available. It is equally preferable . A preferred pressure is 1 kg / cm 2 . The filtration time can be appropriately changed according to the filtration area and the amount of filtrate. Further, it is preferable to add about 1 to 100 g (per 1 L of liquid amount) of filter aid. An example of the filter aid is diatomaceous earth.
[0014]
b. Filter press The filter press comprises a filtering device and a filtering material. As the filtration device, a pressure filtration type, a compression type, a gas blow type, an electroosmotic type, a combination thereof, and the like are known. Moreover, a commercial item can be used for the filtration apparatus. Specifically, a filtration device (trade name ORION C40, manufactured by SEITZ) that combines pressure filtration and gas blow is exemplified.
[0015]
As the filter medium (which is a concept including a filter cloth), fibers (eg, cellulose and the like), resins, inorganic filter aids (diatomaceous earth, pearlite, and the like) are known. A commercial item can be used for this filter medium. Specifically, a filter medium (Zeta Plus LA series manufactured by Cuno Co., Ltd.) that combines highly purified cellulose fibers and acid-treated diatomaceous earth is exemplified. In the following (2) to (4), a filter press similar to the above can be used.
[0016]
(2) Separation of the II + III precipitate fraction and the same supernatant fraction The I supernatant fraction is treated with an ethanol concentration of 18-30%, preferably 20-25%, and separated into a supernatant fraction and a precipitate fraction. To do. Immunoglobulins and the like can be recovered from the precipitated fraction.
1. Examples of ethanol treatment conditions are pH 4.5 to 8 (especially pH 5.2 to 7), temperature 0 to −7 ° C. (particularly −5 to −6 ° C.), and 30 minutes to 15 hours (particularly 1 to 5 hours). .
[0017]
2. Filtration conditions: pore size 0. Examples of the filtration treatment include 6 to 4 μm, a filtration area of 20 to 30 m 2 (per 1000 to 5000 L), a pressure of 1 Kg / cm 2 or less, and a filtration time of 10 hours or less . Among these, the most preferable is a filter medium having a pore width of 0.6 to 2 μm (eg, zeta plus 30LA), but a filter medium having a pore size width of 0.9 to 4 μm (eg, zeta plus 10LA) is also available. It is equally preferable . A preferred pressure is 1 kg / cm 2 . A filter aid can also be used.
[0018]
(3) Separation of the IV precipitate fraction and the supernatant fraction The II + III supernatant fraction is treated with an ethanol concentration of 35 to 45% to separate the supernatant fraction and the precipitate fraction. Antithrombin-III, haptoglobin and the like can be recovered from the precipitated fraction.
1. Examples of ethanol treatment conditions are pH 5 to 7 (particularly pH 5.8 to 6.3), temperature 0 to −7 ° C. (particularly −5 to −6 ° C.), and 30 minutes to 15 hours (particularly 1 to 5 hours). .
[0019]
2. Filtration conditions: pore size 0. Examples of the filtration treatment include 4 to 2 μm, a filtration area of 20 to 30 m 2 (per 1000 to 5000 L), a pressure of 1 Kg / cm 2 or less, and a filtration time of 10 hours or less . Among these, the most preferable is a filter medium having a pore diameter width of 0.4 to 1 μm (eg, Zeta Plus 50LA), and the next preferable is a filter medium having a pore diameter width of 0.6 to 2 μm (eg, Zeta Plus). 30LA). A preferred pressure is 1 kg / cm 2 . Further, 1 to 100 g of filter aid (liquid amount per 1L) added. An example of the filter aid is diatomaceous earth.
[0020]
(4) Separation of the V precipitate fraction and the supernatant fraction The IV supernatant fraction is treated with an ethanol concentration of 35 to 45% to separate the supernatant fraction and the precipitate fraction. Albumin and the like can be recovered from the precipitated fraction.
1. Ethanol treatment conditions pH 4 to 5.5 (particularly pH 4.8 to 5.2), temperature 0 to -7 ° C (particularly -5 to -6 ° C), 30 minutes to 15 hours (particularly 1 to 5 hours) Is done.
[0021]
2. Filtration conditions: pore size 0. Examples of the filtration treatment include 6 to 4 μm, a filtration area of 20 to 30 m 2 (per 1000 to 5000 L), a pressure of 1 Kg / cm 2 or less, and a filtration time of 10 hours or less . Among these, the most preferable is a filter medium having a pore width of 0.6 to 2 μm (eg, zeta plus 30LA), but a filter medium having a pore size width of 0.9 to 4 μm (eg, zeta plus 10LA) is also available. It is equally preferable . A preferred pressure is 1 kg / cm 2 . A filter aid can also be used. The plasma protein thus fractionated can be isolated and purified by a known technique (see JP-A-3-128398).
[0022]
【The invention's effect】
According to the method of the present invention, plasma protein can be efficiently fractionated from a plasma protein-containing material, particularly plasma. Moreover, compared with the conventional centrifugation method, the working environment can be improved (danger, noise, etc.), the plasma fractionation ability can be enhanced, and the work system can be improved (labor and work hours).
[0023]
【Example】
Examples and experimental examples will be given to describe the present invention in more detail, but the present invention is not limited to these examples.
Example 1
Ethanol previously cooled to −20 ° C. was added to 2000 L of the supernatant fraction after separating cryopaste, prothrombin and blood coagulation factor IX from 2000 L of plasma to a final concentration of 8%. The mixture was allowed to stand at pH 7.2 and at a temperature of −2 ° C. for 2 hours to produce a precipitate (fraction I). Using Zeta Plus 30LA (pore size 0.6-2 μm, manufactured by Cuno Co., Ltd.), filtration was performed under the conditions of a filtration area of 12 m 2 and a filtration pressure of 1 kg / cm 2 or less. As a filter aid, 10 g (per 1 L of liquid volume) of acid-treated celite 535 manufactured by Manville was used. The filtration apparatus used was a product name ORION C40 (total length 2920 mm × width 720 mm × height 980 mm) manufactured by SEIZT. The filtration process was completed in 3 hours. The precipitated fraction is a raw material for the fibrinogen preparation. The supernatant fraction was used in the next example.
[0024]
Example 2
Ethanol previously cooled to −20 ° C. was added to 2300 L of the first supernatant fraction to a final concentration of 21%. The mixture was allowed to stand at pH 6.8 at a temperature of −5 ° C. for 2 hours to produce a precipitate (fraction II + III). Using Zeta Plus 30LA, filtration was performed under the conditions of a filtration area of 24 m 2 and a filtration pressure of 1 kg / cm 2 or less. The filtration process was completed in 9 hours. The precipitated fraction is a raw material for the globulin preparation. The supernatant fraction was used in the next example.
[0025]
Example 3
Ethanol previously cooled to −30 ° C. was added to 2700 L of the II + III fraction to a final concentration of 40%. The mixture was allowed to stand at pH 6.0 and at a temperature of −5 ° C. for 2 hours to form a precipitate (Fourth IV fraction). Using Zeta Plus 50LA (pore diameter 0.4-1 μm, manufactured by Cuno Co., Ltd.), filtration was performed under conditions of a filtration area of 16 m 2 and a filtration pressure of 1 kg / cm 2 or less. As a filter aid, 20 g (per 1 L of liquid volume) of acid-treated Celite 535 manufactured by Manville was used. The filtration process was completed in 8 hours. The precipitated fraction is a raw material for the antithrombin-III preparation and the haptoglobin preparation. The IV supernatant fraction was used in the next example.
[0026]
Example 4
4000 L of the IV supernatant fraction (final concentration of ethanol 40%) was adjusted to pH 4.8 and allowed to stand at a temperature of −5 ° C. for 2 hours to produce a precipitate (Fraction V). Using Zeta Plus 30LA, filtration was performed under the conditions of a filtration area of 24 m 2 and a filtration pressure of 1 kg / cm 2 or less. The filtration process was completed in 9 hours. The precipitated fraction is a raw material for the albumin preparation.
[0027]
Example 5
Using the fraction after antithrombin-III was separated from the II + III precipitate fraction of Example 3, filtration was performed according to Example 3. The prepared precipitate fraction becomes a raw material of the haptoglobin preparation.
Experimental example 1 (processing time)
The same starting materials as in the method of the present invention (filtration method) in Example 1 were used in the same amount, and each fraction corresponding to Examples 1 to 4 was converted into a conventional method (sharpless centrifuge (eg, product). The time required for separating and recovering the supernatant fraction and the precipitate fraction by centrifugal separation method using the name Sharpless No. 6A) was compared and examined. The conventional ethanol treatment conditions were the same as in the examples.
[0028]
The results are shown in Table 1.
[0029]
[Table 1]
Experimental Example 2 (Yield comparison)
In the same manner as in Experimental Example 1, the amount of plasma protein that can be recovered by a conventional method from the precipitated fractions (paste) prepared by the method of the present invention (filtration method) and the conventional method (centrifugation method) was compared. The amount was expressed as a converted value per 1000 L of plasma. The results are shown in Table 2. In Table 2, “1 times” of antithrombin-III in the IV fraction corresponds to the amount of antithrombin-III contained in 1 ml of normal human plasma.
[0031]
[Table 2]
Claims (9)
第Ｉ上清画分をエタノール濃度１８〜３０％、ｐＨ４．５〜８の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積２０〜３０ｍThe first supernatant fraction was subjected to low-temperature fractionation treatment under conditions of ethanol concentration 18-30% and pH 4.5-8, pore size 0.6-4 μm, filtration area 20-30 m 22 （液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ(Per 1000-5000L of liquid), pressure 1Kg / cm 22 以下の条件下に濾過処理を行うことにより第ＩＩ＋ＩＩＩ沈殿画分および第ＩＩ＋ＩＩＩ上清画分を分離回収し、By performing filtration under the following conditions, the II + III precipitate fraction and the II + III supernatant fraction are separated and recovered,
第ＩＩ＋ＩＩＩ上清画分をエタノール濃度３５〜４５％、ｐＨ５〜７の条件下での低温分画処理後に孔径０．４〜２μｍ、濾過面積１０〜２０ｍThe II + III supernatant fraction was subjected to low-temperature fractionation treatment under the conditions of ethanol concentration of 35 to 45% and pH of 5 to 7, and the pore size was 0.4 to 2 μm and the filtration area was 10 to 20 m. 22 （液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ(Per 1000-5000L of liquid), pressure 1Kg / cm 22 以下、濾過助剤を１〜１００ｇ（液量１Ｌ当たり）添加の条件下に濾過処理を行うことにより第ＩＶ沈殿画分および第ＩＶ上清画分を分離回収し、Hereinafter, the IV precipitate fraction and the IV supernatant fraction are separated and recovered by performing filtration under the condition of adding 1 to 100 g of filter aid (per 1 L of liquid volume),
第ＩＶ上清画分をエタノール濃度３５〜４５％、ｐＨ４〜５．５の条件下での低温分画処理後に孔径０．６〜４μｍ、濾過面積２０〜３０ｍThe IV supernatant fraction was subjected to low-temperature fractionation under conditions of ethanol concentration of 35 to 45% and pH of 4 to 5.5, and a pore size of 0.6 to 4 μm and a filtration area of 20 to 30 m. 22 （液量１０００〜５０００Ｌ当たり）、圧力１Ｋｇ／ｃｍ(Per 1000-5000L of liquid), pressure 1Kg / cm 22 以下に濾過処理を行うことにより第Ｖ沈殿画分および第Ｖ上清画分を分離回収することを特徴とする血漿蛋白の分画方法。A method for fractionating plasma protein, which comprises separating and recovering the V precipitate fraction and the V supernatant fraction by performing a filtration treatment below.
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| CN102786577A (en) * | 2012-07-20 | 2012-11-21 | 中国医学科学院输血研究所 | Simple device for preparing plasma protein products through batch adsorption, and method thereof |
| US9534029B2 (en) | 2012-10-03 | 2017-01-03 | Csl Behring Ag | Method of purifying proteins |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010459B (en) * | 2010-09-10 | 2012-08-29 | 合肥工业大学 | Method for preparing animal blood plasma albumen powder |
| JP7020615B2 (en) * | 2018-03-30 | 2022-02-16 | 三井化学株式会社 | A method for producing a cell-treated product containing a nitrile hydratase and a method for producing an amide compound. |
-
1993
- 1993-12-17 JP JP34323193A patent/JP3560066B2/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102786577A (en) * | 2012-07-20 | 2012-11-21 | 中国医学科学院输血研究所 | Simple device for preparing plasma protein products through batch adsorption, and method thereof |
| CN102786577B (en) * | 2012-07-20 | 2014-07-09 | 中国医学科学院输血研究所 | Simple device for preparing plasma protein products through batch adsorption, and method thereof |
| US9534029B2 (en) | 2012-10-03 | 2017-01-03 | Csl Behring Ag | Method of purifying proteins |
| US9937229B2 (en) | 2012-10-03 | 2018-04-10 | Csl Behring Ag | Methods of treatment using hemopexin compositions |
| US10918696B2 (en) | 2012-10-03 | 2021-02-16 | Csl Behring Ag | Methods of treatment using hemopexin compositions |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07173190A (en) | 1995-07-11 |
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