JPH01258700A - Purification of blood coagulation ix factor - Google Patents
Purification of blood coagulation ix factorInfo
- Publication number
- JPH01258700A JPH01258700A JP8445888A JP8445888A JPH01258700A JP H01258700 A JPH01258700 A JP H01258700A JP 8445888 A JP8445888 A JP 8445888A JP 8445888 A JP8445888 A JP 8445888A JP H01258700 A JPH01258700 A JP H01258700A
- Authority
- JP
- Japan
- Prior art keywords
- factor
- blood coagulation
- aqueous solution
- antibody
- guanidine
- 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.)
- Pending
Links
- 230000023555 blood coagulation Effects 0.000 title abstract description 5
- 238000000746 purification Methods 0.000 title description 2
- 239000007864 aqueous solution Substances 0.000 claims abstract description 19
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 claims abstract description 12
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 claims abstract description 6
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 claims abstract description 6
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 claims abstract description 6
- 229940116357 potassium thiocyanate Drugs 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 19
- 239000003114 blood coagulation factor Substances 0.000 claims description 7
- 229940019700 blood coagulation factors Drugs 0.000 claims description 6
- 108010076282 Factor IX Proteins 0.000 claims 3
- 102000013831 Coagulation factor IX Human genes 0.000 claims 1
- 238000007670 refining Methods 0.000 claims 1
- 239000000872 buffer Substances 0.000 abstract description 5
- 230000015271 coagulation Effects 0.000 abstract description 4
- 238000005345 coagulation Methods 0.000 abstract description 4
- 229920002684 Sepharose Polymers 0.000 abstract description 3
- 102100022641 Coagulation factor IX Human genes 0.000 abstract description 2
- 239000007983 Tris buffer Substances 0.000 abstract description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 abstract description 2
- 208000009429 hemophilia B Diseases 0.000 abstract description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 abstract description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract 3
- 239000008280 blood Substances 0.000 abstract 3
- 239000004202 carbamide Substances 0.000 abstract 2
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 abstract 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 108010014173 Factor X Proteins 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000011282 treatment Methods 0.000 description 5
- 239000003480 eluent Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 229960004198 guanidine Drugs 0.000 description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000003163 cell fusion method Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102100033806 Alpha-protein kinase 3 Human genes 0.000 description 1
- 101710082399 Alpha-protein kinase 3 Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102100026735 Coagulation factor VIII Human genes 0.000 description 1
- 102100029117 Coagulation factor X Human genes 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 201000003542 Factor VIII deficiency Diseases 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 208000009292 Hemophilia A Diseases 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 208000015294 blood coagulation disease Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 238000007820 coagulation assay 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 compound [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
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229940024788 profilnine Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は純度の高い血液凝固第X因子(以下、第X因子
ともいう)の製造法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing highly pure blood coagulation factor X (hereinafter also referred to as factor X).
より詳細には、血液凝固第X因子含有水溶液を固定化抗
血液凝固第X因子抗体により処理することによる血液凝
固第X因子の精製方法における改良に関する。More specifically, the present invention relates to an improvement in a method for purifying blood coagulation factor X by treating an aqueous solution containing blood coagulation factor X with an immobilized anti-blood coagulation factor X antibody.
第X因子は、通常単独で使用されることはなく、たとえ
ばヒト血液凝固因子因子、第■因子、および第X因子等
を含んだ、所謂第X因子複合体として使用される。Factor X is usually not used alone, but is used as a so-called factor
血液凝固は約20種の促進因子および抑制因子の関連す
るCa5cadeから成り、これらの血液凝固因子の減
少、増大は血液凝固障害を起こし、種々の疾患をもたら
す、それらの疾患のうち、第■因子の欠乏に基づ(もの
を血友病A、第X因子の欠乏に基づくものを血友病Bと
言う。Blood coagulation consists of about 20 promoting factors and inhibitory factors related to Ca5cade, and decreases and increases in these blood coagulation factors cause blood coagulation disorders, resulting in various diseases. Hemophilia A is based on a deficiency of factor X, and hemophilia B is based on a deficiency of factor X.
この第X因子は、臨床的には
i)血友病B患者に対する第X因子の補充、ii)血友
病A患者(第■因子抗体保有者)に対するバイパス療法
、
1ii)凝固因子生合成不全症(薬物投与による)に対
する凝固因子補充療法
などに用いられる。Clinically, this factor X is used in i) supplementation of factor It is used for coagulation factor replacement therapy, etc., for patients with cancer (through drug administration).
本発明の目的は第X因子含有水溶液中に含まれる夾雑物
質を分離除去し、純度の高い安全な第X因子製剤を提供
することにある。An object of the present invention is to separate and remove contaminants contained in a factor X-containing aqueous solution to provide a highly pure and safe factor X preparation.
〔課題を解決するための手段]
上記目的を達成するために本発明者らは種々研究を重ね
て来た結果、第X因子含有水溶液を固定化抗血液凝固第
X因子抗体によって精製処理する際に、溶出液としてチ
オシアン酸カリウムおよびグアニジンから選ばれる少な
くとも一種を含有する水溶液を使用することによって効
率よく、かつ純度高く第X因子が得られることを見出し
、さらに研究を重ねて本発明を完成させるに至った。[Means for Solving the Problems] In order to achieve the above object, the present inventors have conducted various studies and found that when a factor X-containing aqueous solution is purified using an immobilized anti-blood coagulation factor They discovered that factor X could be obtained efficiently and with high purity by using an aqueous solution containing at least one selected from potassium thiocyanate and guanidine as an eluent, and through further research they completed the present invention. reached.
即ち、本発明は第X因子含有水溶液を固定化抗血液凝固
第X因子抗体により処理することによる第X因子の精製
方法において、溶出液が、チオシアン酸カリウムおよび
グアニジンから選ばれる少なくとも一種を含有する水?
8aであることを特徴とする精製方法である。That is, the present invention provides a method for purifying factor X by treating a factor X-containing aqueous solution with an immobilized anti-blood coagulation factor water?
8a.
(a)本発明による処理対象物
本発明の方法の処理対象物は、第■因子含有水ン容液で
ある。(a) Object to be treated according to the present invention The object to be treated in the method of the present invention is an aqueous solution containing factor (2).
第X因子はヒト血漿由来のものであれば特に限定されな
い。ここに第X因子含有水溶液とは、活性成分として第
X因子のみを含有するものの他、たとえば血液分画技術
等によって得られる所の第X因子および他の成分を含む
、所謂第X因子複合体含有溶液であってもよい。Factor X is not particularly limited as long as it is derived from human plasma. The factor X-containing aqueous solution herein refers to a so-called factor It may also be a containing solution.
第X因子複合体は、第X因子を主成分とし、さらに他の
成分、たとえば他の血液凝固に関与する成分を含むもの
をいい、たとえばヒト血液凝固第■因子、第■因子およ
び/または第X因子をも含んでおり、生物学的製剤基準
で規定される規格に適合したものがあげられる。その組
成比は、たとえば1バイアル(20m)当たりに換算す
ると、第X因子、第■因子、第■因子、第X因子を、各
々400〜500単位、100〜500単位、5〜15
0単位、100〜500単位の値で含むものである。Factor It also contains factor X and complies with the standards stipulated by the Biological Products Standards. The composition ratio is, for example, 400 to 500 units, 100 to 500 units, and 5 to 15 units of factor
It includes values of 0 units and 100 to 500 units.
第X因子含有水溶液としては、血漿、血漿分画、血漿濃
縮物などが例示される。Examples of factor X-containing aqueous solutions include plasma, plasma fractions, and plasma concentrates.
(b)固定化抗体による処理
第X因子は抗第■因子抗体よりなる吸着剤、具体的には
固定化抗第■因子抗体を用いたアフィニティークロマト
グラフィーにより高度精製する。(b) Treatment with immobilized antibody Factor
担体としては、ポリクローナル抗体カラム、モノクロー
ナル抗体カラムのどちらを用いてもよい。As the carrier, either a polyclonal antibody column or a monoclonal antibody column may be used.
ポリクローナル抗体に関して、抗第■因子抗体は、高度
に精製した第X因子を動物に免疫し、得られた血清から
回収・精製することによって得られる。Regarding polyclonal antibodies, anti-factor (I) antibodies can be obtained by immunizing animals with highly purified factor X and collecting and purifying the resulting serum.
当該抗血清の製造は公知の方法にて行なえばよく、例え
ば高度精製第X因子とフロイントの完全アジュバントの
混合乳液を作り、動物の皮肉に2〜3回注射し、最終免
疫の数日後採血を行ない室温で凝固せしめた後、4°C
で一夜放置し、3.00Orpm 、20分間の遠心分
離により当該抗血清が得られる。The antiserum may be produced by a known method; for example, a mixed emulsion of highly purified factor After solidifying at room temperature, heat at 4°C.
The antiserum was obtained by centrifugation at 3.00 rpm for 20 minutes.
免疫に用いる動物としては、特に動物種を選ぶ必要はな
く、例えば、ラット、マウス、ウサギ、ヤギ、ウマ等が
挙げられる。当該抗血清の精製は、例えば、J、 Am
、 Chew、 Soc、、 JL2.3386 (1
940)。There is no need to select a particular animal species as the animal used for immunization, and examples include rats, mice, rabbits, goats, horses, and the like. Purification of the antiserum can be carried out, for example, by J. Am.
, Chew, Soc,, JL2.3386 (1
940).
Fed、 Proc、、 Ll、 1161 (195
B)に記載の方法にて行なわれる。Fed, Proc., Ll., 1161 (195
It is carried out by the method described in B).
モノクローナル抗体に関して、抗第■囚子抗体は細胞融
合法によって得られる。細胞融合法は自体既知の手段に
て行なわれ、その−例は増殖性を持った細胞と目的とす
る抗体を産生しているリンパ球とをポリエチレングリコ
ールの存在下で反応せしめることにより、増殖性と抗体
産生能とを同時に兼ねそなえた細胞を製し、この細胞に
抗体を産生せしめるものであり、当該抗体は一個の抗原
決定基に対してのみ反応する単一の抗体である。With respect to monoclonal antibodies, anti-Prison II antibodies are obtained by cell fusion methods. The cell fusion method is carried out by means known per se. For example, by reacting proliferative cells with lymphocytes producing the target antibody in the presence of polyethylene glycol, proliferative This method involves producing cells that have both the ability to produce antibodies and the ability to produce antibodies at the same time, and causing these cells to produce antibodies, which are single antibodies that react only with one antigenic determinant.
より具体的には増殖性を持つ細胞としてマウスミエロー
マ細胞を、抗体産生リンパ球として第X因子で免疫され
たマウス肺臓細胞(B細胞)を用いて融合させ、さらに
目的とする抗体を産生じている細胞をスクリーニングし
て、当該細胞から第X因子のモノクローナル抗体を得る
。More specifically, mouse myeloma cells as proliferative cells are fused with mouse lung cells (B cells) immunized with factor X as antibody-producing lymphocytes, and the desired antibody is further produced. A factor X monoclonal antibody is obtained from the cells.
また、このようにして得られた抗第■因子抗体を、その
活性を失うことなく固定化する方法としては、以下の不
溶性マトリックスを応用する方法をあげることができる
。かかる方法としては、例えばアミノ酸のコポリマーを
使用する方法(J。Further, as a method for immobilizing the anti-factor (I) antibody thus obtained without losing its activity, the following method using an insoluble matrix can be cited. Such methods include, for example, methods using copolymers of amino acids (J.
Biol、 Chem、、236 1970 (196
1)) 、セルロースを使用する方法(Nature、
189.576 (1961)) 、アガロースあ
るいはセファデックスを使用する方法(Nature、
2iiL1491 (1967)、 Nature、
245.3059(1970)) 、ポリアクリルアミ
ドを使用する方法(Bioche+++、、 8.40
74 (1966))などがあげられる。Biol, Chem, 236 1970 (196
1)) Method using cellulose (Nature,
189.576 (1961)), a method using agarose or Sephadex (Nature,
2iiL1491 (1967), Nature,
245.3059 (1970)), method using polyacrylamide (Bioche+++, 8.40
74 (1966)).
これらの方法により抗第■因子抗体を効率良く固定化し
うる。また、このようにして得られた吸着剤を用いるこ
とにより、収率良く、しかも高純度の第X因子を得るこ
とができる。Anti-factor (I) antibodies can be efficiently immobilized by these methods. Furthermore, by using the adsorbent thus obtained, factor X can be obtained in good yield and with high purity.
本発明に関して、固定化抗血液凝固第■因子抗体による
処理は、例えば以下の通りにして行われる。まず、適度
に精製した第■因子含有水溶液を適当な溶媒〔例えば、
0.01〜0.1 M )リス緩衝1(pH6〜8)〕
で平衡化した固定化抗第■因子抗体にアプライする。同
じ溶媒で洗浄後、溶出液で溶出させた第■因子画分を回
収する。Regarding the present invention, treatment with immobilized anti-blood coagulation factor (I) antibodies is carried out, for example, as follows. First, a moderately purified aqueous solution containing factor
0.01-0.1 M) Liss buffer 1 (pH 6-8)]
Apply to immobilized anti-factor ■ antibody equilibrated with After washing with the same solvent, the factor Ⅰ fraction eluted with the eluate is collected.
本発明において溶出液としては、チオシアン酸カリウム
およびグアニジンから選ばれる少なくとも一種を含有す
る水溶液(通常、緩衝液溶液)が使用され、この点が本
発明の特徴点である。In the present invention, an aqueous solution (usually a buffer solution) containing at least one selected from potassium thiocyanate and guanidine is used as the eluent, and this point is a feature of the present invention.
本発明において使用されるチオシアン酸カリウムおよび
グアニジンの濃度は1〜5M、好ましくは2〜4Mであ
る。当該水溶液のpi(は通常6〜8、好ましくは7程
度である。当該溶出液としては、具体的には1〜5Mチ
オシアン酸カリウム含有0.01〜0.1 M )リス
緩衝液(pH6〜8)、1〜5Mグアニジン−塩酸含有
0.01〜0.1 M )リス緩衝液(pH6〜8)な
どが示される。The concentration of potassium thiocyanate and guanidine used in the present invention is 1-5M, preferably 2-4M. The pi of the aqueous solution is usually 6 to 8, preferably about 7. Specifically, the eluent is a 0.01 to 0.1 M lithium buffer solution (pH 6 to 5) containing 1 to 5 M potassium thiocyanate. 8), 1-5M guanidine-hydrochloric acid containing 0.01-0.1M) Lith buffer (pH 6-8), and the like.
本発明の方法においては、さらに塩を除去するために、
所望によりたとえば透析を行う。In the method of the present invention, in order to further remove salts,
For example, dialysis is performed if desired.
(d)製剤化
上記処理に続いて、第X因子を主成分とする医薬品製造
における常套手段に従って処理を行って、極めて安全性
の高い第■因子製剤を提供することができる。(d) Formulation Following the above-mentioned treatments, a highly safe factor (I) preparation can be provided by performing treatments in accordance with conventional methods for manufacturing pharmaceuticals containing factor X as a main component.
本発明の処理による第X因子ないしは第■因子複合体の
回収率は極めて高く、生物学的製剤基阜が規定する規格
にも十二分に適合できるものであ従って、本発明は純度
の高い第■因子製剤を製造する上で、橿めて有効な方法
である。The recovery rate of factor X or factor This is an extremely effective method for producing factor Ⅰ preparations.
本発明をより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って何ら限定される
ものではない。EXAMPLES Examples and experimental examples are given to explain the present invention in more detail, but the present invention is not limited thereto.
なお、第X因子の力価は一段階による凝固測定法(Ne
w Engl、 Med、、 L125〜130 (1
962))によった。The factor X titer was determined by the one-step coagulation assay (Ne
w Engl, Med, L125~130 (1
962)).
実験例1(溶出液の種類)
各種溶出液を用いた場合の、第X因子の回収率と比活性
を検討した(第1表)。Experimental Example 1 (Types of eluates) The recovery rate and specific activity of factor X were investigated using various eluates (Table 1).
第1表
実験例2(′a度)
溶出液としてグアニジン−塩酸含有0.05 M トリ
ス緩衝液(pH7,0)を用いて、各濃度における回収
率と比活性を検討した(第2表)。Table 1 Experimental Example 2 ('a degrees) Using 0.05 M Tris buffer (pH 7.0) containing guanidine-hydrochloric acid as the eluent, the recovery rate and specific activity at each concentration were investigated (Table 2) .
第2表
実施例
(1) 固定化抗体の調製
抗第■因子モノクローナル抗体をCNBr法により活性
化したセファロース4B(ファルマシア社製)に結合さ
せた。Table 2 Example (1) Preparation of immobilized antibody Anti-Factor Ⅰ monoclonal antibody was bound to Sepharose 4B (manufactured by Pharmacia) activated by the CNBr method.
調製した抗第■因子モノクローナル抗体−セファロース
4 B (IgG: 8.6 mg/ ml ofゲル
)は、ゲルl ml当たり第X因子を150 U (1
7U/mg ofIgG)を結合しているものである。The prepared anti-factor II monoclonal antibody-Sepharose 4 B (IgG: 8.6 mg/ml of gel) contained 150 U (1
7U/mg of IgG).
(2)処理方法
瓢 上記で得た固定化抗体1dを0.05 M )リ
ス緩衝n、 (pH7,0)で平衡化した。(2) Treatment method The immobilized antibody 1d obtained above was equilibrated with 0.05 M) Lys buffer (pH 7.0).
このカラムに第■因子含有溶液(profilnine
”比活性2.51J/Ass。)をアプライし、上記の
溶媒で洗浄した。A factor II-containing solution (profilnine) was added to this column.
"Specific activity 2.51 J/Ass.)" was applied and washed with the above solvent.
その後、4Mグアニジン−塩酸含有0.05 M )リ
ス緩衝液(pH1,0>により溶出した。Thereafter, it was eluted with a 4M guanidine-hydrochloric acid-containing 0.05M) Lys buffer (pH 1.0).
(a) 精製した第X因子は、5DS−PAGE法に
より分析したところ、非還元条件下で分子置駒6500
0の単一ピークを示した。(a) Purified factor
It showed a single peak of 0.
(b) 精製した第X因子を用いてフィブリノゲン凝
固時間を測定したところ、3.8時間(第X因子25U
/d)であった。(b) When fibrinogen clotting time was measured using purified factor
/d).
(C) 結合した第X因子は4M−塩酸グアニジンに
よりその約80%が回収された。。(C) About 80% of the bound factor X was recovered by 4M-guanidine hydrochloride. .
(d) 溶出された第X因子の比活性は約90 丁I
t A!116で、その純度は95%以上(SO5−P
AGE)でありtまた、このものは生物学的製剤基準活
性凝固否η試験にも適合した。(d) The specific activity of the eluted factor X is approximately 90%.
tA! 116, and its purity is over 95% (SO5-P
AGE) and also passed the biological product standard activated coagulation test.
特許出願人 株式会社 ミドリ+1Patent applicant Midori +1 Co., Ltd.
Claims (1)
子抗体により処理することによる血液凝固第IX因子の精
製方法において、溶出液がチオシアン酸カリウムおよび
グアニジンから選ばれる少なくとも一種を含有する水溶
液であることを特徴とする精製方法。In a method for purifying blood coagulation factor IX by treating an aqueous solution containing blood coagulation factor IX with an immobilized anti-blood coagulation factor IX antibody, the eluate is an aqueous solution containing at least one selected from potassium thiocyanate and guanidine. A refining method characterized by:
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8445888A JPH01258700A (en) | 1988-04-05 | 1988-04-05 | Purification of blood coagulation ix factor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8445888A JPH01258700A (en) | 1988-04-05 | 1988-04-05 | Purification of blood coagulation ix factor |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH01258700A true JPH01258700A (en) | 1989-10-16 |
Family
ID=13831184
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP8445888A Pending JPH01258700A (en) | 1988-04-05 | 1988-04-05 | Purification of blood coagulation ix factor |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH01258700A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100245542B1 (en) * | 1991-08-22 | 2000-02-15 | 모저 하., 라우페 하. 페. | Process for the purification of factor xiii or xiiia and monoclonal antibody against factor xiiia |
US6280729B1 (en) | 1991-03-01 | 2001-08-28 | Aventis Behring Llc | Preparation of factor IX |
-
1988
- 1988-04-05 JP JP8445888A patent/JPH01258700A/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6280729B1 (en) | 1991-03-01 | 2001-08-28 | Aventis Behring Llc | Preparation of factor IX |
KR100245542B1 (en) * | 1991-08-22 | 2000-02-15 | 모저 하., 라우페 하. 페. | Process for the purification of factor xiii or xiiia and monoclonal antibody against factor xiiia |
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