JPH01275600A - Method for purifying antithrombin-iii - Google Patents
Method for purifying antithrombin-iiiInfo
- Publication number
- JPH01275600A JPH01275600A JP63105783A JP10578388A JPH01275600A JP H01275600 A JPH01275600 A JP H01275600A JP 63105783 A JP63105783 A JP 63105783A JP 10578388 A JP10578388 A JP 10578388A JP H01275600 A JPH01275600 A JP H01275600A
- Authority
- JP
- Japan
- Prior art keywords
- antithrombin
- iii
- aqueous solution
- solution containing
- carrier
- 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.)
- Granted
Links
- 102000004411 Antithrombin III Human genes 0.000 title claims abstract description 19
- 108090000935 Antithrombin III Proteins 0.000 title claims abstract description 19
- 229960005348 antithrombin iii Drugs 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims description 32
- 239000007864 aqueous solution Substances 0.000 claims abstract description 16
- 125000001165 hydrophobic group Chemical group 0.000 claims abstract description 7
- 238000000746 purification Methods 0.000 claims abstract description 6
- 239000003446 ligand Substances 0.000 claims abstract description 5
- 150000003839 salts Chemical class 0.000 claims abstract description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 abstract description 13
- 229920002684 Sepharose Polymers 0.000 abstract description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 abstract description 2
- 206010059245 Angiopathy Diseases 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 239000002510 pyrogen Substances 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 230000000694 effects Effects 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000000356 contaminant Substances 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 4
- 238000002523 gelfiltration Methods 0.000 description 4
- 229920002554 vinyl polymer Polymers 0.000 description 4
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 3
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 3
- 102000004506 Blood Proteins Human genes 0.000 description 3
- 108010017384 Blood Proteins Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108090000190 Thrombin Proteins 0.000 description 3
- 239000003114 blood coagulation factor Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 229960004072 thrombin Drugs 0.000 description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000004019 antithrombin Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229940019700 blood coagulation factors Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- VCUFZILGIRCDQQ-KRWDZBQOSA-N N-[[(5S)-2-oxo-3-(2-oxo-3H-1,3-benzoxazol-6-yl)-1,3-oxazolidin-5-yl]methyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C1O[C@H](CN1C1=CC2=C(NC(O2)=O)C=C1)CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F VCUFZILGIRCDQQ-KRWDZBQOSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical class [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011046 pyrogen test Methods 0.000 description 1
- 238000012958 reprocessing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000006496 vascular abnormality Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000002747 voluntary effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野コ
本発明は、疎水性クロマトを利用したアンチトロンビン
−IIIの精製方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for purifying antithrombin-III using hydrophobic chromatography.
[従来の技術]
アンチトロンビン−■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65,00
0〜68,000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。[Prior art] Antithrombin-■ is a type of glycoprotein belonging to α2 globulin present in plasma, and its molecular weight is 65,000.
0 to 68,000, has protease inhibitory activity, and strongly inhibits thrombin coagulation activity.
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■因子など
に対する阻害作用をも有している。In addition, it has an inhibitory effect not only on thrombin but also on other coagulation factors, such as activated factor X and activated factor II.
その他、プラスミンやトリプシンに対す3阻害作用のあ
ることも報告されている。In addition, it has been reported that it has a 3-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 coexistence of heparin.
このような薬理作用を有するアンチトロンビン−■は、
凝固異常亢進の補正、具体的には汎発性血管異常症(D
I C)を目的として用いられるものである。Antithrombin-■, which has such pharmacological effects,
Correction of hypercoagulopathy, specifically generalized vascular abnormality (D
It is used for the purpose of IC).
[発明が解決しようとする課8]
ところで、アンチトロンビン−IIIの精製工程におい
て、パイロジエ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。[Problem to be Solved by the Invention 8] By the way, in the purification process of antithrombin-III, there is a concern that pyrodiene or contaminant proteins may be mixed in, and various methods for removing them are being studied.
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ために行う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エンの除去等にはあまり有効でな
いことが判明している。Furthermore, the presence of heat-denatured proteins produced by liquid heat treatment at 60° C. for 10 hours to inactivate hepatitis viruses and the like that may be present in plasma protein components is becoming a new problem. Recently, as a method for removing this 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 pyrogenes, etc.
そこで本発明者らはこれらの事情に鑑み、種々研究を重
ねた結果、アンチトロンビン−■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−■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 conducted various studies and found that by treating an aqueous solution containing antithrombin-■ with a hydrophobic chromatographic carrier, the desired purpose was achieved and an antithrombin with superior safety was obtained. It was discovered that a thrombin-■ preparation could be prepared, and the present invention was completed.
[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−■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-■ used in the present invention is not particularly limited as long as it is of human origin and purified to the extent that it can be used as a medicine. For example, It can be purified from human whole blood, plasma, serum, serum compressed from coagulated blood, and the like.
アンチトロンビン−■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−1、IV−4または■−1+IV−4、
クエン酸含有血漿から血液凝固第■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-■ include, for example, plasma, fractions IV-1, IV-4 or IV-1+IV-4 obtained by Cohn's cold ethanol method,
The residual fraction etc. after recovering blood coagulation factor (I) from citric acid-containing plasma are 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5つ一7693
号公報に開示の方法が例示される。As a method for purifying antithrombin-III, for example,
Japanese Unexamined Patent Publication No. 48-35017, Japanese Patent Publication No. 5-7693
The method disclosed in the above publication is 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、−膜内には高度精製品の方が効果が大である。Furthermore, although the purity of antithrombin-III is not particularly limited, a highly purified product is more effective within the membrane.
アンチトロンビン−■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。Antithrombin-■The protein concentration of the aqueous solution containing:
An example is about 0.1 to 10 (W/V)%.
また、アンチトロンビン−■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。In addition, the aqueous solution containing antithrombin-■ is subjected to heat treatment (50 to 70°C,
It is preferable to carry out the test for 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 a hydrophobic group, an alkyl group (about 1 to 10 carbon atoms,
(preferably about 3 to 5), a phenyl group which may have a substituent, and the like.
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 insoluble carriers include cellulose, agarose, dextran, polyacrylamide, amino acid copolymers, polyvinyl-based, polystyrene-based, and the like.
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
これに準する方法によって容易に製造することができる
。Insoluble carriers with hydrophobic groups as ligands include alkyl-type Sepharose (e.g., octyl-type Sepharose, etc.), phenyl-type Sepharose, alkyl-type polyvinyl (
For example, butyl type polyvinyl etc.) are commercially available,
Products other than these can also be easily produced by the same method as commercially available products or a method analogous thereto.
本発明における精製工程は、一般にアンチトロンビン−
■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。The purification step in the present invention generally involves antithrombin-
(2) This is carried out by bringing the containing aqueous solution into contact with an insoluble carrier having a hydrophobic group as a ligand, and the method may be either a column method or a batch method.
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、3
M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 contact conditions include a pH of about 6 to 9 and a salt concentration of about 1 to 5M. Solvents that meet these conditions include 3
20mM sodium citrate buffer (pH 7.5) containing M sodium chloride, 50mM containing 2M sodium chloride
Examples include phosphate buffer (pH 7.5).
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−■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1ν!
に対して該水溶液1〜100 if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 a batch method is used, an aqueous solution containing antithrombin-1 adjusted to the above conditions is brought into contact with the hydrophobic chromatographic carrier equilibrated under the same conditions. The conditions are that the carrier 1ν!
Using the aqueous solution 1-100 if, 2-20℃
An example of this method is to mix the mixture for about 30 minutes to 2 hours. Thereafter, the supernatant is collected 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−■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 using the column method, an aqueous solution containing antithrombin-■ adjusted to the above conditions is developed on the hydrophobic chromatographic carrier equilibrated under the same conditions and packed in a column,
Collect the non-adsorbed fraction.
こうして得られたアンチトロンビン−■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。Antithrombin-1 thus obtained is further purified, if necessary, and then formulated by 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−■含有
水溶液に含まれるパイロジエン、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−■製剤を提供す
ることができる。[Effects of the Invention] According to the purification method of the present invention, it is possible to effectively remove pyrogienes, contaminant proteins, heat-denatured proteins generated by heat treatment, etc. contained in the aqueous solution containing antithrombin-■, thereby improving safety. An excellent antithrombin-■ preparation can be provided.
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。In addition, the process is simple and the method is suitable for mass production, making it 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] Examples will be given to explain the present invention in more detail, but the present invention is not limited to these in any way.
実施例1
コーンの冷アルコール分画法で得られた画分■−1のペ
ースト10kgを生理食塩水100gに懸濁し、硫酸バ
リウムを5(ν/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。Example 1 10 kg of paste of fraction ■-1 obtained by Cohn's cold alcohol fractionation method was suspended in 100 g of physiological saline, barium sulfate was added to give a concentration of 5 (ν/V)%, and the mixture was stirred at room temperature for 30 kg.
The mixture was stirred for a minute, and the trace amount of prothrombin present was removed by adsorption with barium sulfate.
この上澄液をpH6,5に調整し、ポリエチレンク+)
ニア −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+J エチL/ ンク!J :7−ル#4.OOOヲ
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2fl
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−■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。This supernatant was adjusted to pH 6.5 and
Niall #4.000 was added to a concentration of 13 (W/V)%, the resulting precipitate was removed by centrifugation, and then the mixture was added. J:7-ru #4. OOO was added at a concentration of 30 (W/V)%, and the resulting precipitate was collected by centrifugation. Dissolve this precipitate in approximately 2 fl of cold physiological saline.
The solution was injected into a heparin-Sepharose column that had been previously adjusted with physiological saline, and antithrombin-■ was adsorbed onto the column. After washing this column with a 0.4M sodium chloride solution to remove impure proteins, a 2.0M sodium chloride solution was passed through the column and the eluted portion was collected.
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20 m M )を添加し、pH7,5に調整した
。一方、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)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■
製)にアンチトロンビン−■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。Sodium citrate was added to the aqueous solution of antithrombin-III at a concentration of 0.6M to adjust the pH to 7.8, followed by heat treatment at 60°C for 10 hours. Sodium chloride (final concentration 20mM) was added to adjust the pH to 7.5. On the other hand, a butyl-type polyvinyl carrier (butyl-Toyopearl 650, Toyo Soda
After contacting the aqueous solution containing antithrombin-■ (manufactured by Alumni Co., Ltd.), the solution was developed with the above buffer and the unadsorbed fraction was collected.
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。Subsequently, antithrombin-III was concentrated by dialysis against 0.9% sodium chloride solution overnight.
W/V)% aqueous solution was obtained and, if necessary, filtered or centrifuged to obtain a clear liquid.
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水希釈し、IN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−で除菌濾過し、500単位つづ分注し、凍
結乾燥を行って乾燥製剤とした。To this 1 (W/V)% aqueous solution of antithrombin-III, 2 (W/V)% mannitol and 0.2 (W/V)% sodium citrate were added, and 0.2 (W/V)% sodium chloride was added.
After diluting with a small amount of cold distilled water to a concentration of 5% and adjusting the pH to 7.6 with IN sodium hydroxide, sterilization filtering was performed using a sterilized Millipore filter, and 500 units were dispensed and freeze-dried. It was made into a dry formulation.
実施例2
画分IV−1ペーストの代わりに、画分■−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−■製剤を得た。Example 2 Instead of fraction IV-1 paste, fraction ■-1+IV-
All treatments were carried out in accordance with Example 1, except that No. 4 was used, and the same antithrombin-■ preparation as in Example 1 was obtained.
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■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−■製剤を得た。Example 3 All treatments were carried out in accordance with Example 1, except that instead of Fraction IV-1 paste, the residual fraction after treating citric acid-containing plasma at low temperature to recover blood coagulation factor (I) was used. An antithrombin-■ preparation similar to that in Example 1 was obtained.
実験例1
(1)パイロジエンの除去
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−■含有画分中のパイロジエンを
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 Pyrodiene According to Example 1, hydrophobic chromatography was performed, and two tests were conducted to examine pyrodiene in the antithrombin-■ containing fractions before and after the treatment (first sample and (referred to as the second sample). The test method was based on the pyrogen test method listed in the Japanese Pharmacopoeia. The results are shown in Table 1.
第1表
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、パイロジエンが十分に除去されて
いることが判明した。Table 1 As shown in Table 1 above, it was found that the hydrophobic chromatography treatment lowered the total temperature and sufficiently removed pyrodiene.
(2)夾雑蛋白の除去
(ω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−■含有画分中の総蛋白量及びアン
チトロンビン−■活性を測定し、比活性を算出した。総
蛋白量は280nIllの吸光度により測定した。また
、アンチトロンビン−■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I If
中に含まれるアンチトロンビン−■量に相当する。(2) Removal of contaminant proteins (ω specific activity. Perform hydrophobic chromatography according to Example 1, measure the total protein amount and antithrombin-■ activity in the antithrombin-■ containing fraction before and after, and compare The activity was calculated.The total protein amount was measured by absorbance at 280 nIll.Antithrombin-■ activity (AT-III activity) was determined by reacting thrombin with the sample at 28°C for 5 minutes, and adding fibrin to this. The amount of prolongation of clotting time is measured, and the titer is calculated from a pre-prepared calibration curve.However, 1 unit is normal human plasma I If
Corresponds to the amount of antithrombin contained in -■.
得られた結果を第2表に示す。The results obtained are shown in Table 2.
第2表
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, by hydrophobic chromatography,
An increase in AT-III activity and specific activity was observed, and in particular, the specific activity increased from 4.9 (units/mg protein) to 6°4 (units/mg protein).
mg protein).
山)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−■含有画分中に夾雑する血漿蛋白
の有無をオフタロニー法により調べた。その結果を第3
表に示す。1) Removal of contaminant plasma proteins Hydrophobic chromatography was carried out according to Example 1, and the presence or absence of contaminant plasma proteins in the antithrombin-①-containing fractions before and after the treatment was examined by the Ophthalony method. The result is the third
Shown in the table.
第3表
(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−■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。Table 3 (C) Gel filtration Hydrophobic chromatography was performed according to Example 1, and the antithrombin-■ containing fractions before and after the treatment were subjected to gel filtration.
The elution patterns were compared. The results are shown in FIG. Note that 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(pH6,8)
′検出性:280rvの吸光度
第1図に示されるように、処理前の両分は二峰性を示す
。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、−峰性と
なることが判明した。Carrier: TSK Gel G3000SW-XL (manufactured by Tosoh Corporation) Eluent: 0.1M phosphate buffer containing 0.2M sodium chloride (pH 6,8) Detectability: Absorbance at 280rv As shown in Figure 1 , both parts before treatment show bimodality. Among these, the high molecular fraction is considered to be based on heat-denatured proteins, judging from the elution pattern before heat treatment. It was found that this polymer fraction disappeared by hydrophobic chromatography, resulting in a negative peak.
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溜
水に溶解し、5匹のマウスに40.000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 an amount equivalent to 40,000 units/kg was administered to five mice via the tail vein, and observation was continued for 7 days.
No abnormality was observed. Furthermore, when the same solution was administered to rabbits at a dose of 5,000 units/kg and observed for 24 hours, no abnormalities in body temperature were observed.
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−■含有画分のゲル濾過溶出パターンを示す。
特許出願人 株式会社 ミドリ十字
第1図
処 理 前 処 理 後保持時
間(分) 保持時間(分)
手 続 補 正 書(自発)
特許庁長官 吉 1)文 毅 殿
1 事件の表示
昭和63年 特 許願第105783号2 発明の名称
アンチトロンビン−IIIの精製方法
3 補正をする者
(平成元年2月13日住居表示変更)
名称 株式会社 ミトリ十字
代表者須山忠和
5 補正命令の日付(自発)
6 補正により増加する請求項の数
なし
8 補正の内容
(1) 明細書第9頁第15行の
「つづ」を「ずつ」に訂正する。
(2同書第13頁第15行の
r40.000Jをr4.000 Jに訂正する。
以上FIG. 1 shows the gel filtration elution pattern of the antithrombin-■ containing fraction before and after hydrophobic chromatography treatment. Patent applicant Green Juji Co., Ltd. Figure 1 Processing Pre-processing Post-retention time (minutes) Retention time (minutes) Procedure Amendment (spontaneous) Commissioner of the Patent Office Yoshi 1) Moon Yi 1 Indication of the case 1988 Patent Application No. 105783 2 Name of the invention Method for purifying antithrombin-III 3 Person making the amendment (change of address on February 13, 1989) Name Mitri Cross Co., Ltd. Representative Tadakazu Suyama 5 Date of amendment order (voluntary) 6. There is no increase in the number of claims due to the amendment. 8. Contents of the amendment (1) "Zuzu" on page 9, line 15 of the specification is corrected to "zuzu". (2 Correct r40.000J on page 13, line 15 of the same book to r4.000J.
Claims (4)
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) Antithrombin-III, which is characterized by bringing an aqueous solution containing antithrombin-III into contact with an insoluble carrier having a hydrophobic group as a ligand, and collecting the unadsorbed fraction.
Purification method.
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), wherein the contact conditions are pH 6 to 9 and salt concentration 1 to 5M.
アンチトロンビン−IIIの精製方法。(3) The method for purifying antithrombin-III according to claim (1), wherein the hydrophobic group is an alkyl group.
ある請求項(1)記載のアンチトロンビン−IIIの精製
方法。(4) The method for purifying antithrombin-III according to claim (1), wherein the antithrombin-III is heat-treated.
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 |
EP95108077A EP0682949A1 (en) | 1988-04-28 | 1989-04-25 | Virus inactivated antithrombin-III preparation free from pyrogen and thermally denatured protein |
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. |
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 true JPH01275600A (en) | 1989-11-06 |
JP2729484B2 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 |
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Country | Link |
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JP (1) | JP2729484B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004056870A3 (en) * | 2002-12-19 | 2004-10-14 | Octapharma Ag | Method for separation of antithrombin |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5210416A (en) * | 1975-07-09 | 1977-01-26 | Kabi Ab | Composition having compatibility to hepatisis virus and removal of said virus |
JPS52102414A (en) * | 1976-02-25 | 1977-08-27 | Puribenteibu Shisuteimuzu Inc | Removement of endotoxin from biological fluid |
US4259448A (en) * | 1978-01-03 | 1981-03-31 | Nippon Soda Company, Ltd. | Protein adsorbent and process for the purification of urokinase |
JPS57203015A (en) * | 1981-06-10 | 1982-12-13 | Dai Ichi Pure Chem Co Ltd | Purifying method of human urinary callicrein |
JPS5944320A (en) * | 1982-07-30 | 1984-03-12 | ベ−リングヴエルケ・アクチエンゲゼルシヤフト | Preparation of c1 inactivating agent |
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 (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5210416A (en) * | 1975-07-09 | 1977-01-26 | Kabi Ab | Composition having compatibility to hepatisis virus and removal of said virus |
JPS52102414A (en) * | 1976-02-25 | 1977-08-27 | Puribenteibu Shisuteimuzu Inc | Removement of endotoxin from biological fluid |
US4259448A (en) * | 1978-01-03 | 1981-03-31 | Nippon Soda Company, Ltd. | Protein adsorbent and process for the purification of urokinase |
JPS57203015A (en) * | 1981-06-10 | 1982-12-13 | Dai Ichi Pure Chem Co Ltd | Purifying method of human urinary callicrein |
JPS5944320A (en) * | 1982-07-30 | 1984-03-12 | ベ−リングヴエルケ・アクチエンゲゼルシヤフト | Preparation of c1 inactivating agent |
JPS6156133A (en) * | 1984-08-24 | 1986-03-20 | Chemo Sero Therapeut Res Inst | Removal of pyrogens |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004056870A3 (en) * | 2002-12-19 | 2004-10-14 | Octapharma Ag | Method for separation of antithrombin |
Also Published As
Publication number | Publication date |
---|---|
JP2729484B2 (en) | 1998-03-18 |
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