JPS6236487B2 - - Google Patents
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- Publication number
- JPS6236487B2 JPS6236487B2 JP55070632A JP7063280A JPS6236487B2 JP S6236487 B2 JPS6236487 B2 JP S6236487B2 JP 55070632 A JP55070632 A JP 55070632A JP 7063280 A JP7063280 A JP 7063280A JP S6236487 B2 JPS6236487 B2 JP S6236487B2
- Authority
- JP
- Japan
- Prior art keywords
- factor
- ammonium sulfate
- aqueous solution
- fractionation
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 17
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 17
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 238000005194 fractionation Methods 0.000 claims description 14
- 239000007864 aqueous solution Substances 0.000 claims description 13
- 239000000356 contaminant Substances 0.000 claims description 5
- 102000015081 Blood Coagulation Factors Human genes 0.000 claims description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 claims description 2
- 239000003114 blood coagulation factor Substances 0.000 claims description 2
- 229940019700 blood coagulation factors Drugs 0.000 claims description 2
- 239000012503 blood component Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 229950003499 fibrin Drugs 0.000 description 9
- 102000009123 Fibrin Human genes 0.000 description 8
- 108010073385 Fibrin Proteins 0.000 description 8
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 102000006395 Globulins Human genes 0.000 description 7
- 108010044091 Globulins Proteins 0.000 description 7
- 210000002826 placenta Anatomy 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 6
- 230000029663 wound healing Effects 0.000 description 5
- 238000004132 cross linking Methods 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 208000007536 Thrombosis Diseases 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 206010010356 Congenital anomaly Diseases 0.000 description 2
- 208000014763 coagulation protein disease Diseases 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920001983 poloxamer Polymers 0.000 description 2
- 229920001515 polyalkylene glycol Polymers 0.000 description 2
- -1 polyoxyethylene Polymers 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- IYLLULUTZPKQBW-UHFFFAOYSA-N Acrinol Chemical compound CC(O)C(O)=O.C1=C(N)C=CC2=C(N)C3=CC(OCC)=CC=C3N=C21 IYLLULUTZPKQBW-UHFFFAOYSA-N 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241000252095 Congridae Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000603 anti-haemophilic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000002682 general surgery Methods 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Description
本発明は、ヒト血液凝固第因子（以下第
因子という）の精製法に関する。
近年止血血栓機構との関連における創傷治癒機
転の一端として、フイブリノーゲン−フイブリン
系の不溶性フイブリン形成に関与している第
因子の作用が注目されてきた。
先天性血液凝固第因子欠損症で観察される
出血と創傷治癒遅延という異常症状は、第因
子の基質となるフイブリンのγ（ガンマー）錯間
の架橋形成（cross−linking クロスリンキン
グ）不全により起こることが知られている。そし
てまた、フイブリンのγ（ガンマー）鎖間の架橋
形成（cross−linking）のみならず、α（アルフ
ア）鎖間のクロスリンキングの有する生物学的意
義についても関心が向けられるようになつた。す
なわち、前者は、γ（ガンマー）鎖が化学的安定
性を通して尿素に不溶性にするのに対して、後者
はフイブリンの物理的・機械的強固さを規制する
のではないかと考えられるようになつた。さらに
また、フイブリノーゲン精製時ならびに抗血友病
因子精製時などに終始つきまとう夾雑物としてし
か、これまでに把握されていなかつたコールド
インソリユブル グロブリン（cold insoluble
globulin）がフイブリン以外に第因子の基質
となり得て、生物学的にきわめて重要な役割を演
じているらしいことがわかつてきた。創傷治癒の
初期には、フイブリンのα（アルフア）鎖間のク
ロスリンキングが、単にフイブリン間のみなら
ず、コールド イソリユブル グロブリンとの間
においても進行する、という形で、創傷治癒初期
相が第因子に依存しているのではないかと考
えられている。
文献：
ピサノ J.J（Pisano、J.J）、アナルズオブ
ザ ニユーヨーク アカデミー オブ サイ
エンス（Ann.N.Y.Acad.Sci.）、202、98
（1972）
マクドナハ R.P.（McDonagh、R.P.）、ザ
シツクステイーンス インターナシヨナルコ
ングレンス オブ ヘマトロジイ（The 16th
International Congr.Hematol.）、Kyoto、1976
モツシヤー D.F.（Mosher、D.F.）、ジヤ
ーナル オブ バイオロジカル ケミストリー
（J.Biol.Chem.）、250、6614（1975）
以上のことから、第因子製剤は臨床適用に
おいて、先天性ないし後天性血液凝固第因子
欠損症及び減少症はもちろんのこと、広く一般外
科手術後の創傷治癒促進に対して効を奏すること
は明らかである。
第因子の製造原料としては、価格、収量、
既製品の製造との関連性などから、人胎盤を用い
ることが優れており、第因子の製造法として
は、胎盤から硫安分画法を主体にした方法（特開
昭53−59018号）、胎盤由来の粗グロブリン画分か
らポリアルキレングリコール分画法による方法
（特開昭53−136996号）、胎盤抽出物からのリバノ
ール、硫安分画法による方法（特開昭47−13446
号、特開昭47−13447号）などの方法がある。
本発明の目的は硫安分画法による第因子の
精製において、夾雑物をより選択的に除去し、第
因子の効率的な純度の上昇と回収をはかるた
めの方法を提供し、この方法を従来法の硫安分画
工程にかえて、また従来の硫安分画工程、ポリエ
チレングリコール分画工程、その他の分画工程の
付加的工程として利用せしめようとするものであ
り、これによつてより高度に精製された第因
子を得んとするものである。
本発明は、第因子及び夾雑物として他の血
液成分を含有する水溶液を硫安分画法によつて精
製するに際して、該水溶液の処理温度を１゜〜15
℃とすること、該水溶液のPHを6.0〜6.6とするこ
と、該水溶液に添加する硫酸アンモニウムの飽和
度を30〜35％とすること、かかる条件下で処理後
沈澱する画分を回収することによる第因子の
精製方法に関する。
本発明を以下において説明する。
本発明において精製の対象とされるのは第
因子を含有する水溶液であり、たとえばヒト胎
盤、血液その他第因子含有物からの水（たと
えば生理的食塩水、リン酸緩衝液など）抽出物な
どが対象とされる。
本発明においては、かかる抽出物をそのまま、
あるいは従来提案されている分画法（硫安、アク
リノール、ポリアルキレングリコール等による分
画法）を漸次組み合せて適用し、予備的に精製し
たものを用いてもよい。
本発明の硫安分画精製処理がなされる前に第
因子含有水溶液は、蛋白濃度（夾雑蛋白をも含
む）が、１〜３％の水溶液に調整されていること
が好ましく、またその比活性は0.1〜１単位／ml
〔新鮮な正常人血漿１ml中に含まれる第因子
活性量を１単位〔スロンボシス ダイアセシス
ヘモラジカ（Thrombosis et Diathesis
Haemorrhagica）23、455（1970）〕であることが
好ましい。水溶液は、PH6.0〜6.6、好ましくは6.2
〜6.4に調整されていることが必要である。水溶
液の蛋白濃度およびPHの調整には緩衝液たとえば
0.01〜0.1Mのリン酸緩衝液等が使える。
精製処理中の当該水溶液の温度は、通常１゜〜
15℃、好ましくは１゜〜10℃である。添加する硫
酸アンモニウムの飽和度は、30〜35％である。
本発明による硫安分画処理は、前述の条件下で
通常約0.5〜２時間の撹拌後３〜10時間静置する
ことによつて行われる。処理後、第因子は、
たとえば5000〜10000rpmの遠心分離によつて沈
澱として回収される。かくして得られた第因
子画分は、すでに他の方法で十分精製されている
場合は、これを公知の方法に従い透析、除菌ろ
過、凍結乾燥することにより医療用に用いうる第
因子製剤をうる。また、本発明の処理後、第
因子の結晶化あるいは試薬として用いうる程
度に高度精製された第因子を得るためには、
例えば、等電点分画法、特定蛋白の抗体を使つた
アフイニテイークロマトグラフイー等の技術を応
用してもよい。
本発明の精製法によれば、約70〜90％の回収率
が得られ、その比活性の上昇は、前記特定した比
活性の水溶液を用いた場合、精製前にくらべて約
３〜６倍である。また、各種血漿蛋白のウサギ抗
血清を用いた免疫電気泳動法による夾雑蛋白質の
分析によると実施例１で用いた原材料において確
認された夾雑蛋白質が、本発明処理工程をへるこ
とによつて完全に免疫学的に消失している。（表
１）。
次に実施例を挙げて本発明を具体的に説明す
る。
実施例 １
ヒトの胎盤を生理的食塩水をもつて抽出した抽
出液800（胎盤約840個、約510Kg）を遠心分離
し、PHを5.0に調整し生ずる沈澱を遠心分離し、
上清をPH６としてこれに硫酸アンモニウムを25％
飽和になるまで加え、生ずる沈澱を遠心分離によ
り除去、上清に更に硫安をPH７において50％飽和
させることにより粗グロブリン画分を沈澱させて
遠心分離により集めた。この画分を出発原料とし
て本発明の処理をおこなつた。即ち、この出発原
料を0.05Mリン酸緩衝液（PH6.3）に溶解し、蛋
白濃度を２％第因子の比活性を0.7単位／ml
に調整し、その後硫酸アンモニウムを30％飽和に
まで添加し、10℃で約１時間撹拌した。約５時間
静置後、遠心によつて沈澱を回収した。この沈澱
を透析、除菌ろ過、分注を経て凍結乾燥した。総
活性230万単位、蛋白として200ｇを得た。
実施例 ２
実施例１の粗グロブリン画分3.5Kgを、0.005M
のEDTAを含むPH7.5の0.05Mリン酸緩衝液100
に溶解させた。この溶液にプルロニツク（ポリオ
キシエチレンとポリオキシプロピレンの共重合
体：平均分子量15000）7.0Kgを加え溶解させる。
一夜静置して生じた沈澱（グロブリンの精製に供
用できる）を遠心分離して除く。得られた上清に
再びプルロニツクを添加して終濃度25％（Ｗ／
Ｖ）とする。
一夜静置後沈澱を分離し、再び前記緩衝液に溶
かし、蛋白濃度を３％、第因子の比活性を１
単位／mlに調整し、その後硫酸アンモニウムを33
％飽和にまで添加し、５℃で約30分撹拌した。約
２時間静置後、遠心によつて沈澱を回収した。こ
の沈澱を透析、除菌ろ過、分注を経て凍結乾燥し
た。総活性200万単位、蛋白てして180ｇを得た。
The present invention relates to a method for purifying human blood coagulation factor (hereinafter referred to as factor). In recent years, attention has been focused on the action of factor factor, which is involved in the formation of insoluble fibrin in the fibrinogen-fibrin system, as part of the wound healing mechanism in relation to the hemostatic thrombosis mechanism. The abnormal symptoms of bleeding and delayed wound healing observed in congenital blood coagulation factor deficiency are caused by failure of cross-linking between gamma complexes in fibrin, which is the substrate for factor factor. It has been known. Furthermore, interest has been focused not only on cross-linking between fibrin γ (gamma) chains, but also on the biological significance of cross-linking between α (alpha) chains. In other words, it has been thought that the former makes fibrin insoluble in urea through the chemical stability of the γ (gamma) chain, while the latter regulates the physical and mechanical strength of fibrin. . Furthermore, cold chloride, which has so far only been understood as a contaminant that is present throughout the purification of fibrinogen and antihemophilic factors,
Insoluble globulin (cold insoluble)
It has become clear that globulin, other than fibrin, can be a substrate for factor factor and appears to play an extremely important role biologically. In the early stage of wound healing, cross-linking between fibrin alpha chains progresses not only between fibrin but also with cold isosoluble globulin, and the initial phase of wound healing becomes a factor. It is thought that it may be dependent on Literature: Pisano, JJ, Annals of the New York Academy of Science (Ann.NYAcad.Sci.), 202, 98.
(1972) McDonagh, RP, The 16th International Congress of Hematology.
International Congr.Hematol., Kyoto, 1976 Mosher, DF, Journal of Biological Chemistry (J.Biol.Chem.), 250, 6614 (1975). It is clear that it is effective not only for congenital or acquired blood coagulation factor deficiency and deficiency, but also for promoting wound healing after general surgery. The manufacturing raw materials for the first factor include price, yield,
Due to its relevance to the manufacture of ready-made products, it is better to use human placenta, and methods for producing factor factor include methods mainly based on ammonium sulfate fractionation from placenta (Japanese Patent Application Laid-Open No. 53-59018); A method using polyalkylene glycol fractionation from placenta-derived crude globulin fraction (Japanese Patent Application Laid-Open No. 136996/1982), a method using ribanol and ammonium sulfate fractionation from placenta extract (Japanese Patent Application Laid-open No. 13446/1982)
There are methods such as JP-A No. 47-13447). An object of the present invention is to provide a method for more selectively removing impurities and efficiently increasing the purity and recovery of factor in the purification of factor by ammonium sulfate fractionation, and to improve the efficiency of factor It is intended to be used in place of the ammonium sulfate fractionation step of the method, and as an additional step to the conventional ammonium sulfate fractionation step, polyethylene glycol fractionation step, and other fractionation steps. The purpose is to obtain purified factor factor. In the present invention, when purifying an aqueous solution containing factor factor and other blood components as impurities by ammonium sulfate fractionation, the processing temperature of the aqueous solution is set at 1° to 15°.
℃, the pH of the aqueous solution is 6.0 to 6.6, the degree of saturation of ammonium sulfate added to the aqueous solution is 30 to 35%, and the fraction that precipitates after treatment under these conditions is collected. This invention relates to a method for purifying factor. The invention will be explained below. In the present invention, the target of purification is an aqueous solution containing factor, such as an aqueous solution (e.g., physiological saline, phosphate buffer, etc.) from human placenta, blood, or other factor-containing substances. targeted. In the present invention, such an extract is used as it is,
Alternatively, previously proposed fractionation methods (fractionation methods using ammonium sulfate, acrinol, polyalkylene glycol, etc.) may be applied in gradual combinations, and preliminary purified products may be used. Before the ammonium sulfate fractionation and purification treatment of the present invention is carried out, the factor-containing aqueous solution is preferably adjusted to have a protein concentration (including contaminant proteins) of 1 to 3%, and its specific activity is 0.1-1 unit/ml
[1 unit of factor activity contained in 1 ml of fresh normal human plasma [Thrombosis diacesis]
Thrombosis et Diathesis
Haemorrhagica) 23, 455 (1970)]. The aqueous solution has a pH of 6.0 to 6.6, preferably 6.2.
It is necessary that it is adjusted to ~6.4. To adjust the protein concentration and pH of aqueous solutions, use buffers such as
A 0.01-0.1M phosphate buffer can be used. The temperature of the aqueous solution during the purification process is usually 1°~
The temperature is 15°C, preferably 1° to 10°C. The saturation degree of ammonium sulfate added is 30-35%. The ammonium sulfate fractionation treatment according to the present invention is carried out under the above-mentioned conditions, usually by stirring for about 0.5 to 2 hours and then allowing to stand for 3 to 10 hours. After processing, the first factor is
For example, it is recovered as a precipitate by centrifugation at 5,000 to 10,000 rpm. If the factor fraction thus obtained has already been sufficiently purified by other methods, it can be subjected to dialysis, sterile filtration, and freeze-drying according to known methods to obtain a factor preparation that can be used for medical purposes. . In addition, in order to crystallize factor or obtain factor highly purified to the extent that it can be used as a reagent after the treatment of the present invention,
For example, techniques such as isoelectric focusing and affinity chromatography using antibodies for specific proteins may be applied. According to the purification method of the present invention, a recovery rate of about 70 to 90% can be obtained, and when an aqueous solution with the specified specific activity is used, the increase in specific activity is about 3 to 6 times that before purification. It is. Furthermore, analysis of contaminant proteins by immunoelectrophoresis using rabbit antiserum of various plasma proteins revealed that the contaminant proteins identified in the raw materials used in Example 1 were completely removed by undergoing the treatment process of the present invention. It has disappeared immunologically. (Table 1). Next, the present invention will be specifically explained with reference to Examples. Example 1 800 extracts (approximately 840 placentas, approximately 510 kg) of human placenta with physiological saline were centrifuged, the pH was adjusted to 5.0, and the resulting precipitate was centrifuged.
Adjust the supernatant to pH 6 and add 25% ammonium sulfate to it.
The resulting precipitate was removed by centrifugation, and the supernatant was further enriched with ammonium sulfate at pH 7 to achieve 50% saturation to precipitate the crude globulin fraction, which was collected by centrifugation. This fraction was used as a starting material for the treatment of the present invention. That is, this starting material was dissolved in 0.05M phosphate buffer (PH6.3), the protein concentration was 2%, and the specific activity of factor was 0.7 units/ml.
After that, ammonium sulfate was added to 30% saturation, and the mixture was stirred at 10°C for about 1 hour. After standing still for about 5 hours, the precipitate was collected by centrifugation. This precipitate was subjected to dialysis, sterilization filtration, dispensing, and freeze-drying. A total activity of 2.3 million units and 200 g of protein were obtained. Example 2 3.5Kg of the crude globulin fraction of Example 1 was added to 0.005M
100% PH7.5 0.05M phosphate buffer containing EDTA
It was dissolved in Add 7.0 kg of Pluronic (polyoxyethylene and polyoxypropylene copolymer: average molecular weight 15,000) to this solution and dissolve.
Remove the precipitate (which can be used for globulin purification) formed by standing overnight by centrifugation. Pluronic was added again to the obtained supernatant to give a final concentration of 25% (W/
V). After standing overnight, the precipitate was separated and dissolved again in the above buffer solution, the protein concentration was 3%, and the specific activity of factor was 1%.
unit/ml, then add ammonium sulfate to 33
% saturation and stirred at 5°C for about 30 minutes. After standing still for about 2 hours, the precipitate was collected by centrifugation. This precipitate was subjected to dialysis, sterilization filtration, dispensing, and freeze-drying. A total activity of 2 million units and 180 g of protein were obtained.
【表】【table】
Claims (1)
の血液成分を含有する水溶液を硫安分画法によつ
て精製する方法において、水溶液の温度１゜〜15
℃、PH6.0〜6.6、添加する硫酸アンモニウムの飽
和度30〜35％とし、沈澱する画分を回収すること
を特徴とするヒト血液凝固第因子の精製方
法。1 In a method for purifying an aqueous solution containing human blood coagulation factor and other blood components as contaminants by ammonium sulfate fractionation, the temperature of the aqueous solution is 1° to 15°.
℃, pH 6.0 to 6.6, saturation of added ammonium sulfate to 30 to 35%, and collecting a precipitated fraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7063280A JPS56166121A (en) | 1980-05-27 | 1980-05-27 | Purifying method of human blood coagulation factor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7063280A JPS56166121A (en) | 1980-05-27 | 1980-05-27 | Purifying method of human blood coagulation factor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS56166121A JPS56166121A (en) | 1981-12-21 |
| JPS6236487B2 true JPS6236487B2 (en) | 1987-08-07 |
Family
ID=13437201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7063280A Granted JPS56166121A (en) | 1980-05-27 | 1980-05-27 | Purifying method of human blood coagulation factor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS56166121A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58121219A (en) * | 1982-01-11 | 1983-07-19 | Green Cross Corp:The | Production of human blood coagulation eighth factor |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5842623B2 (en) * | 1976-08-09 | 1983-09-21 | 株式会社東芝 | Hybrid IC |
-
1980
- 1980-05-27 JP JP7063280A patent/JPS56166121A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS56166121A (en) | 1981-12-21 |
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