JP2729484B2 - Purification method of antithrombin-III - Google Patents
Purification method of antithrombin-IIIInfo
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- JP2729484B2 JP2729484B2 JP63105783A JP10578388A JP2729484B2 JP 2729484 B2 JP2729484 B2 JP 2729484B2 JP 63105783 A JP63105783 A JP 63105783A JP 10578388 A JP10578388 A JP 10578388A JP 2729484 B2 JP2729484 B2 JP 2729484B2
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- antithrombin
- iii
- fraction
- aqueous solution
- pyrogen
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Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明は、疎水性クロマトを利用したアンチトクロン
ビン−IIIの精製方法に関する。Description: TECHNICAL FIELD The present invention relates to a method for purifying antitoclonbin-III using hydrophobic chromatography.
［従来の技術］ アンチトロンビン−IIIは血漿中に存在するα２グロ
ブリンに属する糖蛋白質の一種で、その分子量は65,000
〜68,000であり、プロテアーゼ阻害活性を有し、トロン
ビンの凝固活性を強く阻害する。The [Prior Art] antithrombin -III a kind of glycoprotein belonging to the alpha 2 globulin present in the plasma, its molecular weight 65,000
It has a protease inhibitory activity and strongly inhibits the clotting activity of thrombin.
また、トロンビンに対する阻害作用のみならず、その
他の凝固因子、例えば、活性化Ｘ因子、活性化IX因子な
どに対する阻害作用をも有している。その他、プラスミ
ンやトリプシンに対する阻害作用のあることも報告され
ている。In addition, it has an inhibitory effect not only on thrombin but also on other coagulation factors, for example, activated factor X, activated factor IX, and the like. In addition, it has been reported that it has an inhibitory effect on plasmin and trypsin.
これらの阻害作用は、一般にヘパリンの共存下でより
速やかに進行することが知られている。It is known that these inhibitory effects generally progress more rapidly in the presence of heparin.
このような薬理作用を有するアンチトロンビン−III
は、凝固異常亢進の補正、具体的には汎発性血管異常症
（DIC）を目的として用いられるものである。Antithrombin-III having such a pharmacological action
Is used for correction of hypercoagulability, specifically for the purpose of generalized vascular abnormalities (DIC).
［発明が解決しようとする課題］ ところで、アンチトロンビン−IIIの精製工程におい
て、パイロジェン又は夾雑蛋白の混在が危惧されてお
り、その除去方法が種々検討されている。また、血漿蛋
白成分に混在する可能性のある肝炎ウィルス等を不活性
化するように行う60℃、10時間の液状加熱処理により生
成する熱変性蛋白の存在も新たな問題となりつつある。
最近、この熱変性蛋白を除去する方法として、固定化ヘ
パリンを用いて再処理する方法が提案されている（特開
昭63−23896号公報）。しかし、この方法はパイロジェ
ンの除去等にはあまり有効でないことが判明している。[Problems to be Solved by the Invention] By the way, in the step of purifying antithrombin-III, there is a concern that pyrogen or contaminant proteins may be present, and various methods for removing the same have been studied. In addition, the presence of heat denatured proteins generated by a liquid heat treatment at 60 ° C. for 10 hours performed to inactivate hepatitis virus and the like which may be present in plasma protein components is also becoming a new problem.
Recently, as a method of removing the heat-denatured protein, a method of reprocessing using immobilized heparin has been proposed (Japanese Patent Laid-Open No. 63-23896). However, it has been found that this method is not very effective for removing pyrogen and the like.
そこで本発明者らはこれらの事情に鑑み、種々検討を
重ねた結果、アンチトロンビン−III含有水溶液を疎水
性クロマト担体で処理することにより、所期の目的を達
成し、安全性により優れたアンチトロンビン−III製剤
を調製できることを見出し、本発明を完成した。In view of these circumstances, the present inventors have conducted various studies, and as a result, by treating an antithrombin-III-containing aqueous solution with a hydrophobic chromatographic carrier, the intended object has been achieved, and an anti-thrombin-III antibacterial agent having better safety has been achieved. The inventors have found that a thrombin-III preparation can be prepared, and have completed the present invention.
［課題を解決するための手段］ 本発明で使用されるアンチトロンビン−IIIは、ヒト
由来のもので、医薬として使用できる程度に精製された
ものであれば特に制限されるものではなく、例えば、ヒ
トの全血、血漿、血清または凝固した血液から圧搾され
た血清等から精製することができる。[Means for Solving the Problems] The antithrombin-III used in the present invention is not particularly limited as long as it is of human origin and is purified to the extent that it can be used as a medicine. It can be purified from human whole blood, plasma, serum or serum expressed from coagulated blood.
アンチトロンビン−IIIを調製するための出発原料と
しては、例えば、血漿、コーンの冷エタノール法で得ら
れる画分IV、IV−１、IV−４またはIV−１＋IV−４、ク
エン酸含有血漿から血液凝固第VIII因子を回収した後の
残渣画分等が使用される。Starting materials for preparing antithrombin-III include, for example, plasma, fractions IV, IV-1, IV-4 or IV-1 + IV-4 obtained by the cold ethanol method of corn, and blood from citrate-containing plasma. The residual fraction after collecting the coagulation factor VIII is used.
アンチトロンビン−IIIの精製法としては、例えば、
特開昭48−35017号公報、特公昭59−7693号公報に開示
の方法が例示される。As a method for purifying antithrombin-III, for example,
The methods disclosed in JP-A-48-35017 and JP-B-59-7693 are exemplified.
また、アンチトロンビン−IIIの純度は特に限定され
ないが、一般的には高度精製品の方が効果が大である。Further, the purity of antithrombin-III is not particularly limited, but generally, highly purified products are more effective.
アンチトロンビン−III含有水溶液の蛋白濃度として
は、0.1〜10（W/V）％程度が例示される。The protein concentration of the antithrombin-III-containing aqueous solution is, for example, about 0.1 to 10 (W / V)%.
また、アンチトロンビン−III含有水溶液は、本発明
の疎水性クロマト処理に付す前に加熱処理（50〜70℃、
５〜30時間程度）を行っておくことが好ましい。Further, the antithrombin-III-containing aqueous solution is subjected to a heat treatment (at 50 to 70 ° C.,
(About 5 to 30 hours).
本発明で用いる疎水性基をリガンドとする不溶性担体
は以下のようなものである。The insoluble carrier having a hydrophobic group as a ligand used in the present invention is as follows.
疎水性基としては、アルキル基（炭素数１〜10程度、
好ましくは３〜５程度）、置換基を有していてもよいフ
ェニル基等が挙げられる。As the hydrophobic group, an alkyl group (about 1 to 10 carbon atoms,
(Preferably about 3 to 5), and a phenyl group which may have a substituent.
不溶性担体としては、セルロース、アガロース、デキ
ストラン、ポリアクリルアミド、アミノ酸共重合体、ポ
リビニル系、ポリスチレン系等が挙げられる。Examples of the insoluble carrier include cellulose, agarose, dextran, polyacrylamide, amino acid copolymer, polyvinyl, and polystyrene.
疎水性基をリガンドとする不溶性担体としては、アル
キル型セファロース（例えば、オクチル型セファロース
等）、フェニル型セファロース、アルキル型ポリビニル
（例えば、ブチル型ポリビニル等）等が市販されてお
り、これら以外のものについても市販品と同様な方法ま
たはこれに準ずる方法によって容易に製造することがで
きる。 本発明における精製工程は、一般にアンチトロ
ンビン−III含有水溶液を、疎水性基をリガンドとする
不溶性担体に接触させることにより行われるが、その方
法としてはカラム法、バッチ法の何れにて行ってもよ
い。Examples of the insoluble carrier having a hydrophobic group as a ligand include alkyl-type sepharose (eg, octyl-type sepharose), phenyl-type sepharose, and alkyl-type polyvinyl (eg, butyl-type polyvinyl), and the like. Can also be easily produced by a method similar to a commercially available product or a method analogous thereto. The purification step in the present invention is generally performed by bringing an antithrombin-III-containing aqueous solution into contact with an insoluble carrier having a hydrophobic group as a ligand, and the method may be performed by any of a column method and a batch method. Good.
接触条件は、pH6〜９程度、塩濃度１〜5M程度が例示
される。このような条件を具備する溶媒としては、3M塩
化ナトリウム含有20mMクエン酸ナトリウム緩衝液（pH7.
5）、2M塩化ナトリウム含有50mMリン酸緩衝液（pH7.5）
等が例示される。Examples of the contact conditions include a pH of about 6 to 9 and a salt concentration of about 1 to 5M. As a solvent satisfying such conditions, a 20 mM sodium citrate buffer containing 3 M sodium chloride (pH 7.
5), 50mM phosphate buffer containing 2M sodium chloride (pH7.5)
Etc. are exemplified.
本発明の精製方法を具体的に説明すると、バッチ法に
て行う場合、上記条件に調整したアンチトロンビン−II
I含有水溶液を、同じ条件で平衡化した当該疎水性クロ
マト用担体に接触させる。その条件としては、該担体1m
lに対して該水溶液１〜100mlを用い、２〜20℃で30分〜
２時間程度混和させる方法が挙げられる。その後に遠心
分離にて上澄を回収する。To specifically explain the purification method of the present invention, when the batch method is used, antithrombin-II adjusted to the above conditions is used.
The aqueous solution containing I is brought into contact with the hydrophobic chromatography carrier equilibrated under the same conditions. The conditions are as follows:
1 to 100 ml of the aqueous solution for 1 to 30 minutes at 2 to 20 ° C.
A method of mixing for about 2 hours can be used. Thereafter, the supernatant is recovered by centrifugation.
カラム法にて行う場合、上記条件に調整したアンチト
ロンビン−III含有水溶液を、同じ条件で平衡化され且
つカラム充填された当該疎水性クロマト用担体にて展開
し、非吸着画分を回収する。When the column method is used, the antithrombin-III-containing aqueous solution adjusted to the above conditions is developed on the hydrophobic chromatography carrier equilibrated under the same conditions and packed in a column, and a non-adsorbed fraction is collected.
こうして得られたアンチトロンビン−IIIは、必要に
応じて、さらに精製した後、公知の方法にて製剤化され
る。The antithrombin-III thus obtained is further purified, if necessary, and then formulated into a known method.
［発明の効果］ 本発明の精製方法によれば、アンチトロンビン−III
含有水溶液に含まれるパイロジェン、夾雑蛋白、加熱処
理により生成した熱変性蛋白等を効果的に除去すること
ができ、安全性に優れたアンチトロンビン−III製剤を
提供することができる。[Effect of the Invention] According to the purification method of the present invention, antithrombin-III
It is possible to effectively remove pyrogen, contaminating proteins, heat-denatured proteins generated by heat treatment, and the like contained in the aqueous solution, and to provide an antithrombin-III preparation excellent in safety.
しかも、処理工程が簡便であり、大量製造にも好適な
方法であるので、工業的製法として極めて有用である。Moreover, since the treatment process is simple and suitable for mass production, it is extremely useful as an industrial production method.
［実施例］ 本発明をより詳細に説明するため実施例を挙げて説明
するが、本発明はこれらによってなんら限定されるもの
ではない。[Examples] The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
実施例１ コーンの冷アルコール分画法で得られた画分IV−１の
ペースト10kgを生理食塩水100lに懸濁し、硫酸バリウム
を５（W/V）％になるように加え、室温で30分間攪拌
し、微量に存在するプロトロンビンを硫酸バリウムに吸
着させて除去した。この上澄液をpH6.5に調整し、ポリ
エチレングリコール＃4,000を13（W/V）％になるように
加え、生じた沈澱を遠心分離して除き、さらにポリエチ
レングリコール＃4,000を30（W/V）％になるように加
え、生じた沈澱を遠心分離して回収した。この沈澱を冷
生理食塩水約20lに溶解し、予め生理食塩水で調整され
たヘパリンセファロースのカラムに注入し、アンチトロ
ンビン−IIIをカラムに吸着させた。このカラムを0.4M
の塩化ナトリウム溶液で洗浄して不純蛋白を除いた後、
2.0Mの塩化ナトリウム溶液をカラムに流して溶出してく
る部分を回収した。Example 1 10 kg of the paste of fraction IV-1 obtained by the cold alcohol fractionation method of corn was suspended in 100 l of physiological saline, and barium sulfate was added to a concentration of 5 (W / V)%. After stirring for a minute, a small amount of prothrombin was adsorbed on barium sulfate and removed. The supernatant was adjusted to pH 6.5, polyethylene glycol # 4,000 was added to 13 (W / V)%, the resulting precipitate was removed by centrifugation, and polyethylene glycol # 4,000 was further added to 30 (W / V). V)%, and the resulting precipitate was collected by centrifugation. This precipitate was dissolved in about 20 L of cold physiological saline, injected into a column of heparin sepharose prepared in advance with physiological saline, and antithrombin-III was adsorbed on the column. 0.4M
After washing with sodium chloride solution to remove impure proteins,
A 2.0 M sodium chloride solution was passed through the column to collect the eluted portion.
このアンチトロンビン−IIIの水溶液にクエン酸ナト
リウムを0.6Mの濃度に加え、pH7.8に調整した後、60℃
で10時間の加熱処理を施した後、塩化ナトリウム（最終
濃度3M）及びクエン酸ナトリウム（最終濃度20mM）を添
加し、pH7.5に調整した。一方、3M塩化ナトリウム含有2
0mMクエン酸ナトリウム緩衝液（pH7.5）で平衡化したブ
チル型ポリビニル系担体（ブチル−トヨパール650、東
洋曹達（株）製）にアンチトロンビン−III含有水溶液
を接触させたのちに上記緩衝液で展開し、未吸着画分を
回収した。続いて、0.9％塩化ナトリウム溶液に対して
一夜透析を行いつつ濃縮してアンチトロンビン−IIIの
１（W/V）％水溶液を得、必要に応じて、濾過又は遠心
分離を行って澄明な液とした。Sodium citrate was added to the aqueous solution of antithrombin-III to a concentration of 0.6 M and adjusted to pH 7.8.
After heating for 10 hours, sodium chloride (final concentration 3M) and sodium citrate (final concentration 20 mM) were added to adjust the pH to 7.5. On the other hand, 3M sodium chloride containing 2
After contacting an aqueous solution containing antithrombin-III with a butyl-type polyvinyl carrier (butyl-Toyopearl 650, manufactured by Toyo Soda Co., Ltd.) equilibrated with 0 mM sodium citrate buffer (pH 7.5), It was developed and the unadsorbed fraction was collected. Subsequently, the solution was concentrated while performing dialysis overnight against a 0.9% sodium chloride solution to obtain a 1% (W / V) aqueous solution of antithrombin-III. If necessary, the solution was filtered or centrifuged to obtain a clear solution. And
このアンチトロンビン−IIIの１（W/V）％水溶液にマ
ンニトール２（W/V）％とクエン酸ナトリウム0.2（W/
V）％を加え、塩化ナトリウムが0.5％になるように少量
の冷蒸留希釈し、1Nの水酸化ナトリウムでpH7.6に調整
した後、滅菌したミリポアフィルターで除筋濾過し、50
0単位ずつ分注し、凍結乾燥を行って乾燥製剤とした。A 1 (W / V)% aqueous solution of antithrombin-III was added to 2 (W / V)% mannitol and 0.2 (W / V) sodium citrate.
V), dilute with a small amount of cold distillate to a sodium chloride concentration of 0.5%, adjust the pH to 7.6 with 1N sodium hydroxide, and remove the muscle with a sterilized Millipore filter.
0 units were dispensed and freeze-dried to obtain a dry preparation.
実施例２ 画分IV−１ペーストの代わりに、画分IV−１＋IV−４
を用いる以外は全て実施例１に準じて処理を行い、実施
例１と同様なアンチトロンビン−III製剤を得た。Example 2 Instead of Fraction IV-1 paste, Fraction IV-1 + IV-4
The treatment was carried out in the same manner as in Example 1 except for using, to obtain the same antithrombin-III preparation as in Example 1.
実施例３ 画分IV−１ペーストの代わりに、クエン酸含有血漿を
低温で処理して血液凝固第VIII因子を回収した後の残渣
画分を用いる以外は全て実施例１に準じて処理を行い、
実施例１と同様なアンチトロンビン−III製剤を得た。Example 3 Instead of the fraction IV-1 paste, the treatment was carried out in the same manner as in Example 1 except that the citrate-containing plasma was treated at a low temperature to collect the blood coagulation factor VIII, and the residual fraction was used. ,
The same antithrombin-III preparation as in Example 1 was obtained.
実験例１ （１）パイロジェンの除去 実施例１に準じて、疎水性クロマト処理を行い、その
前後のアンチトロンビン−III含有画分中のパイロジェ
ンを調べる試験を２回行った（各々第１サンプル及び第
２サンプルと称する）。なお、試験方法は日本薬局方収
載の発熱性物質試験法によった。その結果を第１表に示
す。Experimental Example 1 (1) Removal of pyrogen According to Example 1, a hydrophobic chromatographic treatment was carried out, and a test for examining pyrogen in the antithrombin-III-containing fraction before and after that was performed twice (the first sample and the first sample, respectively). Referred to as a second sample). In addition, the test method was based on the pyrogenic substance test method listed in the Japanese Pharmacopoeia. Table 1 shows the results.
上記第１表に示されるように、疎水性クロマト処理に
より合計温度が低下し、パイロジェンが十分に除去され
ていることが判明した。 As shown in Table 1 above, it was found that the total temperature was lowered by the hydrophobic chromatography treatment, and pyrogen was sufficiently removed.
（２）夾雑蛋白の除去 （ａ）比活性 実施例１に準じて疎水性クロマト処理を行い、その前
後のアンチトロンビン−III含有画分中の総蛋白量及び
アンチトロンビン−III活性を測定し、比活性を算出し
た。総蛋白量は280nmの吸光度により測定した。また、
アンチトロンビン−III活性（AT−III活性）はトロンビ
ンと試料とを28℃で５分間反応させ、これにフィブリン
を加えた時に凝固時間がどの程度延長されるかを測定
し、予め作製しておいた検量線よりその力価を算出した
ものである。但し、１単位は正常人血漿1ml中に含まれ
るアンチトロンビン−III量に相当する。得られた結果
を第２表に示す。(2) Removal of contaminating proteins (a) Specific activity A hydrophobic chromatographic treatment was carried out according to Example 1, and the total protein amount and antithrombin-III activity in the antithrombin-III-containing fraction before and after the treatment were measured. The specific activity was calculated. Total protein was measured by absorbance at 280 nm. Also,
Antithrombin-III activity (AT-III activity) was prepared by reacting thrombin with a sample at 28 ° C. for 5 minutes, measuring how much the coagulation time was prolonged when fibrin was added thereto, and preparing it in advance. The titer was calculated from the calibration curve. However, one unit corresponds to the amount of antithrombin-III contained in 1 ml of normal human plasma. Table 2 shows the obtained results.
第２表に示されるように、疎水性クロマト処理によ
り、AT−III活性及び比活性の上昇が認められ、特に比
活性は4.9（単位/mg蛋白）から6.4（単位/mg蛋白）とな
った。 As shown in Table 2, an increase in AT-III activity and specific activity was observed by the hydrophobic chromatography treatment, and the specific activity was particularly increased from 4.9 (units / mg protein) to 6.4 (units / mg protein). .
（ｂ）夾雑血漿蛋白の除去 実施例１に準じて疎水性クロマト処理を行い、その前
後のアンチトロンビン−III含有画分中に夾雑する血漿
蛋白の有無をオクタロニー法により調べた。その結果を
第３表に示す。(B) Removal of contaminating plasma proteins Hydrophobic chromatography treatment was performed according to Example 1, and the presence or absence of contaminating plasma proteins in the antithrombin-III-containing fraction before and after the treatment was examined by the Ouchterlony method. Table 3 shows the results.
（ｃ）ゲル濾過 実施例１に準じて疎水性クロマト処理を行い、その前
後のアンチトロンビン−III含有画分をゲル濾過にか
け、その溶出パターンを比較した。その結果を第１図に
示す。なお、ゲル濾過の条件は次のとおりである。 (C) Gel Filtration A hydrophobic chromatographic treatment was performed in the same manner as in Example 1, and the antithrombin-III-containing fractions before and after that were subjected to gel filtration, and their elution patterns were compared. The result is shown in FIG. The conditions for gel filtration are as follows.
担体:TSKゲルG3000SW−XL（東ソー社製） 溶出液:0.2M塩化ナトリウム含有0.1Mリン酸緩衝液（pH
6.8） 検出法:280nmの吸光度 第１図に示されるように、処理前の画分は二峰性を示
す。このうち、高分子画分は加熱処理前の溶出パターン
からみて、熱変性蛋白に基づくものと考えられる。疎水
性クロマト処理によりこの高分子画分は消失し、一峰性
となることが判明した。Carrier: TSK gel G3000SW-XL (manufactured by Tosoh Corporation) Eluent: 0.1 M phosphate buffer containing 0.2 M sodium chloride (pH
6.8) Detection method: absorbance at 280 nm As shown in FIG. 1, the fraction before treatment shows bimodality. Among these, the polymer fraction is considered to be based on the heat-denatured protein in view of the elution pattern before the heat treatment. It was found that this high molecular fraction disappeared by the hydrophobic chromatography treatment and became monomodal.
実験例２ アンチトロンビン−IIIの凍結乾燥製剤を注射用蒸留
水に溶解し、５匹のマウスに4,000単位/kg相当量を尾静
脈より投与して７日間観察を続けたが、なんら異常は認
められなかった。また、同じ溶解液を家兎に5,000単位/
kg投与して24時間観察したが、体温の異常は認められな
かった。Experimental Example 2 A freeze-dried preparation of antithrombin-III was dissolved in distilled water for injection, and 4,000 units / kg equivalent was administered to 5 mice via the tail vein and observation was continued for 7 days, but no abnormality was observed. I couldn't. Also, the same lysate was added to rabbits at 5,000 units /
Observation for 24 hours after administration of kg showed no abnormal body temperature.
第１図は、疎水性クロマト処理の前後でのアンチトロン
ビン−III含有画分のゲル濾過溶出パターンを示す。FIG. 1 shows gel filtration elution patterns of antithrombin-III-containing fractions before and after hydrophobic chromatography.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭52−102414（ＪＰ，Ａ) 特開 昭52−10416（ＪＰ，Ａ) 特開 昭59−44320（ＪＰ，Ａ) 特開 昭57−203015（ＪＰ，Ａ) 特開 昭61−56133（ＪＰ，Ａ) 米国特許4259448（ＵＳ，Ａ) ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-52-102414 (JP, A) JP-A-52-10416 (JP, A) JP-A-59-44320 (JP, A) JP-A-57-104 203015 (JP, A) JP-A-61-56133 (JP, A) US Pat. No. 4,259,448 (US, A)
Claims (5)
ビン−III含有水溶液を、疎水性基をリガンドとする不
溶性担体に接触させ、その未吸着画分を回収することを
特徴とするアンチトロンビン−IIIの精製方法。1. An antithrombin-III solution comprising contacting an aqueous solution containing human plasma-derived antithrombin-III which has been subjected to heat treatment with an insoluble carrier having a hydrophobic group as a ligand, and collecting a non-adsorbed fraction thereof. Purification method.
を実質的に吸着除去する条件下で接触させることからな
る請求項（１）記載のアンチトロンビン−IIIの精製方
法。2. The method for purifying antithrombin-III according to claim 1, which comprises contacting at least a condition under which pyrogen and heat-denatured protein are substantially adsorbed and removed.
アルブミン、ハプトグロビン、α２−マクログロブリ
ン、α１−アンチトリプシン及びIgAを実質的に吸着除
去する条件下で接触させることからなる請求項（１）記
載のアンチトロンビン−IIIの精製方法。(3) at least a pyrogen, a heat-denatured protein,
Albumin, haptoglobin, alpha 2 - macroglobulin, alpha 1 - claim (1) method for purifying antithrombin -III according comprising contacting antitrypsin and IgA under conditions that substantially adsorbed and removed.
又は（３）記載のアンチトロンビン−IIIの精製方法。4. The method according to claim 2, wherein the hydrophobic group is an alkyl group.
Or the method for purifying antithrombin-III according to (3).
る請求項（２）又は（３）記載のアンチトロンビン−II
Iの精製方法。5. The antithrombin-II according to (2) or (3), wherein the contact conditions are pH 6-9 and salt concentration 1-5M.
Purification method of I.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63105783A JP2729484B2 (en) | 1988-04-28 | 1988-04-28 | Purification method of antithrombin-III |
| CA000597405A CA1341379C (en) | 1988-04-28 | 1989-04-21 | Purified antithrombin-iii and methods of producing the same |
| DE68925918T DE68925918T2 (en) | 1988-04-28 | 1989-04-25 | Method for purifying antithrombin III and antithrombin III composition containing this antithrombin III |
| EP89304085A EP0339919B1 (en) | 1988-04-28 | 1989-04-25 | Method for purifying antithrombin-III and antithrombin-III preparation containing said antithrombin-III |
| ES89304085T ES2084599T3 (en) | 1988-04-28 | 1989-04-25 | METHOD FOR PURIFYING ANTITROMBINE III AND PREPARATION OF ANTITROMBINE III CONTAINING SUCH ANTITROMBINE III. |
| EP95108077A EP0682949A1 (en) | 1988-04-28 | 1989-04-25 | Virus inactivated antithrombin-III preparation free from pyrogen and thermally denatured protein |
| KR1019890005684A KR0139049B1 (en) | 1988-04-28 | 1989-04-28 | Method for purifying antithrombin-ñ and antithrombin-ñ preparation containing antithrombin-ñ |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63105783A JP2729484B2 (en) | 1988-04-28 | 1988-04-28 | Purification method of antithrombin-III |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01275600A JPH01275600A (en) | 1989-11-06 |
| JP2729484B2 true JP2729484B2 (en) | 1998-03-18 |
Family
ID=14416743
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63105783A Expired - Lifetime JP2729484B2 (en) | 1988-04-28 | 1988-04-28 | Purification method of antithrombin-III |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2729484B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE0203770D0 (en) * | 2002-12-19 | 2002-12-19 | Biovitrum Ab | Method of separation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4259448A (en) | 1978-01-03 | 1981-03-31 | Nippon Soda Company, Ltd. | Protein adsorbent and process for the purification of urokinase |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL49752A (en) * | 1975-07-09 | 1979-07-25 | Kabi Ab | Compositions having affinity for hepatitis virus and method for hepatitis virus removal or concentration |
| JPS52102414A (en) * | 1976-02-25 | 1977-08-27 | Puribenteibu Shisuteimuzu Inc | Removement of endotoxin from biological fluid |
| JPS57203015A (en) * | 1981-06-10 | 1982-12-13 | Dai Ichi Pure Chem Co Ltd | Purifying method of human urinary callicrein |
| DE3228502A1 (en) * | 1982-07-30 | 1984-02-02 | Behringwerke Ag, 3550 Marburg | METHOD FOR PRODUCING THE C1 INACTIVATOR AND ITS USE |
| JPS6156133A (en) * | 1984-08-24 | 1986-03-20 | Chemo Sero Therapeut Res Inst | Removal of pyrogens |
-
1988
- 1988-04-28 JP JP63105783A patent/JP2729484B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4259448A (en) | 1978-01-03 | 1981-03-31 | Nippon Soda Company, Ltd. | Protein adsorbent and process for the purification of urokinase |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01275600A (en) | 1989-11-06 |
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