JPH1059867A - Activation of blood coagulation factor vii and production of activated blood coagulation factor vii by the activation method - Google Patents
Activation of blood coagulation factor vii and production of activated blood coagulation factor vii by the activation methodInfo
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- JPH1059867A JPH1059867A JP8237145A JP23714596A JPH1059867A JP H1059867 A JPH1059867 A JP H1059867A JP 8237145 A JP8237145 A JP 8237145A JP 23714596 A JP23714596 A JP 23714596A JP H1059867 A JPH1059867 A JP H1059867A
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- fvii
- blood coagulation
- coagulation factor
- solution
- factor vii
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本願発明は、血漿蛋白質の分野に
関する。より詳細には、活性化血液凝固第ＶＩＩ因子
(以下、ＦＶＩＩaと称することがある)の製造方法に関
し、ＦＶＩＩを含有する血漿画分または遺伝子組換え技
術に基づいて作出されたＦＶＩＩ産生細胞培養上清か
ら、陰イオン交換クロマトグラフィーによる処理及びそ
れに連続した液相中での活性化によって、夾雑蛋白質含
量が低減されたＦＶＩＩaを、極めて簡便に生産し得る
ＦＶＩＩaの製造方法を提供するものである。The present invention relates to the field of plasma proteins. More specifically, activated blood coagulation factor VII
(Hereinafter, may be referred to as FVIIa), from an FVII-containing plasma fraction or an FVII-producing cell culture supernatant produced based on a gene recombination technique, by anion exchange chromatography and the like. An object of the present invention is to provide a method for producing FVIIa, which can extremely easily produce FVIIa in which the content of contaminant proteins is reduced by activation in a continuous liquid phase.
【０００２】[0002]
【従来の技術並びに発明が解決しようとする課題】ＦＶ
ＩＩはビタミンＫ依存性の血液凝固因子であり、外因系
血液凝固の開始因子であることは広く知られている。他
のビタミンＫ依存性凝固因子と同様にＮ末端から３５残
基までのアミノ酸配列に１０個のγカルボキシグルタミ
ン酸(以下、Ｇｌａと称することがある)からなるＧｌａ
領域を有している(Ｐｒｏｃ.Ｎａｔｌ.Ａｃａｄ.Ｓｃ
ｉ.ＵＳＡ,ｖｏｌ.８３,ｐ.２４１２−２４１６,１９８
６)。ＦＶＩＩは、ｉｎ ｖｉｔｒｏにおいて、活性化血
液凝固第Ｘ因子(以下、ＦＸaと称することがある)、活
性化血液凝固第ＩＸ因子(以下、ＦＩＸaと称することが
ある)またはトロンビン(以下、ＦＩＩaと称することが
ある)によって、152Ａｒｇ−153Ｉｌｅが加水分解さ
れ、一個のＳ−Ｓ結合で架橋されたＨ鎖とＬ鎖から構成
される活性型ＦＶＩＩすなわちＦＶＩＩaに変換される
ことが知られている(Ｊ.Ｂｉｏｌ.Ｃｈｅｍ.,ｖｏｌ.２
５１,ｐ.４７９７−４８０２,１９７６)。また、最近、
Ｓａｕｌｉｕｓ等は生理的なＦＶＩＩ活性化酵素が活性
化血液凝固第Ｘ因子であると報告した(Ｂｉｏｃｈｅｍ
ｉｓｔｒｙ,ｖｏｌ.３５,ｐ.１９０４−１９１０,１９
９６)。2. Description of the Related Art FV
II is a vitamin K-dependent blood coagulation factor and is widely known to be an initiator of extrinsic blood coagulation. Similar to other vitamin K-dependent coagulation factors, Gla consisting of 10 γ-carboxyglutamic acids (hereinafter sometimes referred to as Gla) in the amino acid sequence from the N-terminal to 35 residues.
Region (Proc. Natl. Acad. Sc.
i. USA, vol. 83, p. 2412-2416, 198.
6). In vitro, FVII is activated blood coagulation factor X (hereinafter sometimes referred to as FXa), activated blood coagulation factor IX (hereinafter sometimes referred to as FIXa) or thrombin (hereinafter referred to as FIIa). Is sometimes hydrolyzed to convert it into activated FVII, which is composed of H and L chains cross-linked by one SS bond, ie, FVIIa (J). .Biol.Chem., Vol.2
51, p. 4797-4802, 1976). Also recently,
Saulius et al. Reported that physiological FVII activating enzyme is activated blood coagulation factor X (Biochem).
istry, vol. 35, p. 1904-1910, 19
96).
【０００３】血友病Ａ及び血友病Ｂ患者に対する補充療
法として、血液凝固第ＶIII因子(以下、ＦＶIIIと称す
ることがある)及び血液凝固第ＩＸ因子(以下、ＦＩＸと
称することがある)製剤の投与が行なわれている。しか
し、当該治療法に伴いＦＶIII及びＦＩＸに対する中和
抗体(インヒビターと呼ばれることもある)の出現が問題
視されている。このようなインヒビター患者の治療にＦ
ＶＩＩaが有効であることは既に報告されており、現
在、血漿由来ＦＶＩＩa及び遺伝子組換え技術を応用し
た遺伝子組換え型ＦＶＩＩaの開発が進められている(基
礎と臨床,ｖｏｌ.３０,ｐ.３１５−３２５,１９９６;Ｈ
ａｅｍｏｓｔａｓｉｓ,ｖｏｌ.１９,ｐ.３３５−３４
３,１９８９)[0003] As replacement therapy for hemophilia A and hemophilia B patients, blood coagulation factor VIII (hereinafter sometimes referred to as FVIII) and blood coagulation factor IX (hereinafter sometimes referred to as FIX) preparations Is being administered. However, the appearance of neutralizing antibodies (sometimes called inhibitors) against FVIII and FIX has been regarded as a problem with the treatment. F for the treatment of such inhibitor patients
It has already been reported that VIIa is effective, and currently, the development of plasma-derived FVIIa and recombinant FVIIa applying genetic recombination technology is underway (Basic and Clinical, vol. 30, p. 315). -325, 1996; H
aemostasis, vol.19, p.335-34
3, 1989)
【０００４】酵素前駆体であるＦＶＩＩからＦＶＩＩa
への変換に関しては各種の試験が行なわれ、ＦＶＩＩ活
性化酵素であるＦＸa、ＦＩＸa及びＦＩＩaが存在しな
い場合、ＦＶＩＩaの関与によるＦＶＩＩの自己活性化
反応が生じること、即ち次式で示される反応"ＦＶＩＩ
＋ ＦＶＩＩa → ２ＦＶＩＩa"で進行することが明らか
にされている(Ｂｉｏｃｈｅｍｉｓｔｒｙ,ｖｏｌ.２８,
ｐ.９３３１−９３３６,１９８９)。ところで、ＦＶＩ
Ｉaの調製方法として、陰イオン交換樹脂を使用した活
性化方法がある(Ｒｅｓ.Ｄｉｓｃｌ.,ｖｏｌ.２６９,
ｐ.５６４−５６５,１９８６)。この方法は、反応の詳
細は明らかにされていないが、ＦＶＩＩaを工業規模で
生産するうえで極めて有効な方法である。しかしなが
ら、陰イオン交換樹脂を使用したＦＶＩＩの活性化にお
いて問題となるのは、副産物としてＦＶＩＩのＬ鎖のＮ
末端から３８残基のアミノ酸が欠失したＧｌａ領域欠失
ＦＶＩＩa(以下、ＧＤ-ｌｅｓｓＦＶＩＩaと称すること
がある)が生成することにある。ＦＶＩＩaの生体内での
活性発現にはリン脂質及び組織因子との結合が必要であ
り、Ｇｌａ領域はそのためには必須である。従って、Ｇ
ｌａ領域の存在しないＧＤ-ｌｅｓｓＦＶＩＩaはリン脂
質及び組織因子との結合が極めて弱く、生物学的活性を
有さずその存在の生理的意義はないものと考えられてい
る(Ｊ.Ｂｉｏｌ.Ｃｈｅｍ.,ｖｏｌ.２６５,ｐ.１８９０
−１８９４,１９９０；Ｔｈｒｏｍｂｏｓｉｓ ａｎｄ
Ｈａｅｍｏｓｔａｓｉｓ.,ｖｏｌ.６７,ｐ.６７９−６
８５,１９９２)。[0004] The enzyme precursors FVII to FVIIa
Various tests have been conducted for the conversion to FVII, and in the absence of the FVII activating enzymes FXa, FIXa and FIIa, a self-activation reaction of FVII due to the involvement of FVIIa occurs, ie, a reaction represented by the following formula: FVII
+ FVIIa → 2FVIIa "(Biochemistry, vol. 28,
pp. 9331-9336, 1989). By the way, FVI
As a method for preparing Ia, there is an activation method using an anion exchange resin (Res. Discl., Vol. 269,
564-565, 1986). This method is an extremely effective method for producing FVIIa on an industrial scale, although the details of the reaction are not disclosed. However, the problem with the activation of FVII using anion exchange resins is that the by-product N
This is to produce Gla region-deleted FVIIa in which 38 amino acids have been deleted from the terminal (hereinafter sometimes referred to as GD-lessFVIIa). Expression of FVIIa activity in vivo requires binding to phospholipids and tissue factor, and the Gla region is essential for that. Therefore, G
GD-lessFVIIa having no la region has a very weak binding to phospholipids and tissue factor, has no biological activity, and is considered to have no physiological significance (J. Biol. Chem. , vol. 265, p. 1890
-1894, 1990; Thrombosis and
Haemostasis., Vol. 67, p. 679-6.
85, 1992).
【０００５】これまで、陰イオン交換樹脂上での活性化
はゲルにＦＶＩＩを展開した後、完全活性化させ、グラ
ジエント溶出することにより、溶出段階で副産物のＧＤ
-ｌｅｓｓＦＶＩＩaとＦＶＩＩaの等電点の差異を利用
して分離する方法が報告されており、Ｔｈｉｍ等はＭｏ
ｎｏＱを使用した陰イオン交換クロマトグラフィーによ
る活性化反応においては産生されたＧＤ-ｌｅｓｓＦＶ
ＩＩaは分離可能と述べている(Ｂｉｏｃｈｅｍｉｓｔｒ
ｙ,ｖｏｌ.２７,ｐ.７７８５−７７９３,１９８８)。し
かしながら、実際上その完全分離は極めて難しく、分離
を厳密に行なえば工程間収率は著しく低減する。さら
に、これまでに陰イオン交換樹脂上でのＧＤ-ｌｅｓｓ
ＦＶＩＩaの産生機構に関する報告はなく、その制御を
陰イオン交換樹脂上で行なうことは極めて困難であっ
た。Up to now, activation on an anion exchange resin has been achieved by developing FVII on a gel, fully activating the gel, and performing gradient elution, whereby GD of by-products is eluted in the elution step.
-less FVIIa and FVIIa have been reported to use a difference in isoelectric point to separate them.
GD-lessFV produced in the activation reaction by anion exchange chromatography using noQ
IIa states that it is separable (Biochemistr).
y, vol. 27, p. 7785-7793, 1988). However, in practice, complete separation is extremely difficult, and strict separation can significantly reduce the yield between steps. Furthermore, GD-less on anion exchange resin
There has been no report on the mechanism of FVIIa production, and it was extremely difficult to control it on an anion exchange resin.
【０００６】[0006]
【課題を解決するための手段】本願発明者等は活性化反
応における上記問題点に鑑み、陰イオン交換樹脂上での
活性化反応における副産物であるＧＤ-ｌｅｓｓＦＶＩ
Ｉaの産生を抑制するべく鋭意研究を重ね種々の検討を
行なった結果、本願発明を完成するに至った。本願発明
は、陰イオン交換樹脂上における活性化反応を６０％未
満に抑制し、溶出したＦＶＩＩ／ＦＶＩＩa混合溶液を
０.５ｍＭ以上のＣａ2+が存在する溶液中での液相中で
熟成させ９５％以上活性化させる方法をその技術の骨子
とする。本願発明を適用すれば、活性化工程におけるＦ
ＶＩＩからＦＶＩＩaの変換において生ずるＧＤ-ｌｅｓ
ｓＦＶＩＩaの産生が抑制され、著しい工程間収率の上
昇が可能となる。In view of the above-mentioned problems in the activation reaction, the present inventors have considered that GD-lessFVI which is a by-product in the activation reaction on an anion exchange resin.
As a result of intensive studies and various studies to suppress the production of Ia, the present invention was completed. The present invention suppresses the activation reaction on an anion exchange resin to less than 60%, and matures the eluted FVII / FVIIa mixed solution in a liquid phase in a solution containing 0.5 mM or more of Ca 2+. The method of activating 95% or more is the gist of the technology. By applying the present invention, F in the activation step
GD-les generated in the conversion of VII to FVIIa
The production of sFVIIa is suppressed, and the yield between steps can be significantly increased.
【０００７】以下、本願発明の詳細について説明する。
本願発明で用いられる出発物質は、血漿または血漿を
適当なクロマトグラフィー操作またはコーンのエタノー
ル分離法もしくはその改良法を用いて調製されたＦＶＩ
Ｉ含有溶液または、遺伝子組換え技術に基づいて作出
されたＦＶＩＩ産生形質転換細胞より調製されるＦＶＩ
Ｉ及び／もしくはＦＶＩＩ誘導体含有溶液が対象となる
が、最適な出発原料としては、血漿を陰イオンクロマト
グラフィーで粗精製したＧｌａ領域を有する蛋白溶液
(ＰＰＳＢ画分)を抗ＦＶＩＩモノクローナル抗体固定化
アフィニティーゲルに展開し溶出したほぼＦＶＩＩのみ
を含有する溶液である。ＦＶＩＩの精製は特に免疫吸着
の原理に基づく方法に限定されるものではなく、ＦＶＩ
Ｉを純化可能な方法であればいずれでもよい。Hereinafter, the present invention will be described in detail.
The starting material used in the present invention is FVI prepared by using plasma or plasma by an appropriate chromatography operation or a method of separating ethanol from corn or an improved method thereof.
I-containing solution or FVI prepared from an FVII-producing transformed cell produced based on a genetic recombination technique
The most suitable starting material is a protein solution having a Gla region obtained by roughly purifying plasma by anion chromatography.
(PPSB fraction) is a solution containing almost only FVII which was developed and eluted on an affinity gel immobilized with an anti-FVII monoclonal antibody. The purification of FVII is not particularly limited to a method based on the principle of immunoadsorption.
Any method can be used as long as I can be purified.
【０００８】前記ＦＶＩＩ画分を、陰イオン交換樹脂ク
ロマトグラフィーに供する。陰イオン交換樹脂は陰イオ
ン交換樹脂であれば特に限定されるものではないが、好
適にはＤＥＡＥセファロースファーストフロウ(ファル
マシア社)及びＱセファロースファーストフロウ(ファル
マシア社)等が使用され、ＦＶＩＩ画分を樹脂と接触さ
せる。この工程では種々の条件を採用することができ、
上記リガンドとの接触はバッチ法または連続カラム法で
実施することができるが、最適な態様としては、前記の
陰イオン交換樹脂をクロマトグラフィーのカラムに充填
し、試料を通液後、吸着した所望のＦＶＩＩを溶出させ
る。陰イオン交換樹脂からの溶出は０.５ｍＭ以上のＣ
ａ2+を含む緩衝液を用いて段階的溶出法(ステップワイ
ズ溶出法)により、溶出液中のＦＶＩＩ／ＦＶＩＩa混合
溶液のＦＶＩＩaへの活性化率が６０％以下になるよう
に溶出する。溶出液中の活性化率を制御するためには、
陰イオン交換樹脂へのＦＶＩＩ展開量と樹脂内での滞留
時間を調整すればよい。すなわち、樹脂へのＦＶＩＩ展
開量及び樹脂内での滞留時間の増加は活性化率を増大さ
せ、樹脂へのＦＶＩＩ展開量及び樹脂内での滞留時間の
減少は活性化率を低下せしめる。[0008] The FVII fraction is subjected to anion exchange resin chromatography. The anion exchange resin is not particularly limited as long as it is an anion exchange resin. Preferably, DEAE Sepharose Fast Flow (Pharmacia) and Q Sepharose Fast Flow (Pharmacia) are used, and the FVII fraction is used. Contact with resin. In this step, various conditions can be adopted,
The contact with the ligand can be carried out by a batch method or a continuous column method. In an optimal embodiment, the anion exchange resin is packed in a chromatography column, the sample is passed, and the adsorbed resin is adsorbed. Of FVII. Elution from the anion exchange resin is 0.5 mM or more C
The FVII / FVIIa mixed solution in the eluate is eluted by a stepwise elution method (stepwise elution method) using a buffer containing a 2+ so that the activation rate of FVIIa to FVIIa is 60% or less. To control the activation rate in the eluate,
The amount of FVII developed on the anion exchange resin and the residence time in the resin may be adjusted. That is, an increase in the amount of FVII spread to the resin and the residence time in the resin increase the activation rate, and a decrease in the amount of FVII spread to the resin and the residence time in the resin lowers the activation rate.
【０００９】溶出後の熟成時間は、溶出時のＦＶＩＩ
／ＦＶＩＩaの混合比率、ＦＶＩＩ／ＦＶＩＩaの蛋白
濃度によって決定される。溶出液中の蛋白濃度は陰イオ
ン交換樹脂へのＦＶＩＩ展開量及びカラムサイズが決定
されれば一定になる。好適には１.０〜３.０ｍｇ／ｍｌ
が望ましく、そのように陰イオン交換樹脂への展開量及
びカラムサイズを決定する。また、溶出時の活性化率は
陰イオン交換樹脂へのＦＶＩＩ展開量が決定されれば、
上述のごとく樹脂内での滞留時間を制御すればよい。ま
た、熟成開始時のＣａ2+濃度は０.５ｍＭ以上が要求さ
れ、それ以下の濃度ではＧＤ-ｌｅｓｓＦＶＩＩaの産生
が顕著になり、適正ではない。好適には１.０〜１０ｍ
Ｍの濃度範囲が使用される。熟成の終了は、ＦＶＩＩ／
ＦＶＩＩaの混合溶液のＦＶＩＩaの活性化率が９５％を
越えた時点で、希釈操作によって蛋白濃度を減じ活性化
反応速度を遅延させるか、またはｐＨを低下させ酵素反
応を一時停止せしめることによって行なう。得られたＦ
ＶＩＩa溶液は透析操作によって所望の製薬学的調合剤
に処方すればよい。The aging time after elution is determined by the FVII at the time of elution.
/ FVIIa mixing ratio and FVII / FVIIa protein concentration. The protein concentration in the eluate becomes constant if the amount of FVII developed on the anion exchange resin and the column size are determined. Preferably 1.0-3.0 mg / ml
It is desirable to determine the amount of development on the anion exchange resin and the column size. Further, the activation rate at the time of elution is determined if the amount of FVII developed on the anion exchange resin is determined.
The residence time in the resin may be controlled as described above. Further, the Ca 2+ concentration at the start of ripening is required to be 0.5 mM or more, and at a concentration lower than that, GD-lessFVIIa production becomes remarkable and is not appropriate. Preferably 1.0 to 10 m
A concentration range of M is used. End of ripening is FVII /
When the activation rate of FVIIa in the mixed solution of FVIIa exceeds 95%, the protein concentration is reduced by a dilution operation to delay the activation reaction rate, or the enzyme reaction is temporarily stopped by lowering the pH. Obtained F
The VIIa solution may be formulated into a desired pharmaceutical preparation by a dialysis operation.
【００１０】静脈内投与のための調合剤に対しては、組
成物を、通常、生理学的に適合し得る物質例えば塩化ナ
トリウム、グリシン等を含み且つ生理学的条件に適合し
得る緩衝されたｐＨを有する水溶液中に溶解する。ま
た、長期安定性の確保の観点から、最終的剤型として凍
結乾燥製剤の形態を採ることも考慮され得る。なお、静
脈内に投与される組成物のガイドラインは政府の規則、
例えば「生物学的製剤基準」によって確立されている。
以下に、実施例を挙げて本願発明を具体的に説明する
が、本願発明は何等これらに限定されるものではない。For preparations for intravenous administration, the compositions are usually formulated with a buffered pH that includes physiologically compatible substances such as sodium chloride, glycine, and the like, and that is compatible with physiological conditions. Dissolves in the aqueous solution. In addition, from the viewpoint of securing long-term stability, it may be considered to take the form of a lyophilized preparation as the final dosage form. The guidelines for compositions administered intravenously are governed by government regulations,
For example, it has been established by the “Biological Standards”.
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
【００１１】[0011]
【実施例】実施例の記述に先立ち、本願発明において使
用されたＦＶＩＩ活性及びＦＶＩＩa含量の測定方法に
ついて概説する。 １) ＦＶＩＩの生物学的活性 ＦＶＩＩは血液凝固の開始因子である組織因子と結合し
血液凝固を開始する。ＦＶＩＩの定量方法は、検体をＦ
ＶＩＩ欠乏血漿に添加後、一定時間インキュベーション
した後、組織因子、リン脂質及びＣａ2+を含有したＰＴ
試薬を添加しその凝固時間から算出する。 ２) ＦＶＩＩa含量の測定 ＦＶＩＩa含量の測定にはＳＤＳ−ＰＡＧＥを使用す
る。ＦＶＩＩ(分子量５０ｋｄａ)は活性化すると１個の
Ｓ−Ｓ結合で結合した２本鎖に分かれる。Ｈ鎖は分子量
３０ｋｄａ、Ｌ鎖は分子量２０ｋｄａ。還元系のＳＤＳ
−ＰＡＧＥでは未活性体は分子量５０ｋｄａの位置に、
Ｈ鎖は３０ｋｄａ、Ｌ鎖は２０ｋｄａの位置に確認され
る。検出されるバンドをデンシトメーターで読み取り、
分子量５０ｋｄａのバンドを未活性化ＦＶＩＩ含量％、
Ｈ鎖とＬ鎖の含量の和をＦＶＩＩa含量％とした。活性
化率はＦＶＩＩa含量をＦＶＩＩ含量とＦＶＩＩa含量の
和で除した値を百分率(％)で表した。EXAMPLES Prior to the description of the examples, the method of measuring FVII activity and FVIIa content used in the present invention will be outlined. 1) Biological activity of FVII FVII binds to tissue factor which is an initiation factor of blood coagulation and initiates blood coagulation. The method of quantifying FVII is as follows:
After adding to VII-deficient plasma and incubating for a certain period of time, PT containing tissue factor, phospholipid and Ca 2+ was added.
Calculate from the clotting time of the reagent added. 2) Measurement of FVIIa content SDS-PAGE is used for measurement of FVIIa content. When activated, FVII (molecular weight 50 kda) is separated into two strands linked by one SS bond. The H chain has a molecular weight of 30 kda, and the L chain has a molecular weight of 20 kda. Reduction SDS
-In the PAGE, the inactive compound is located at a position of a molecular weight of 50 kda,
The H chain is confirmed at a position of 30 kda, and the L chain is confirmed at a position of 20 kda. Read the detected band with a densitometer,
The band with a molecular weight of 50 kda was converted to the unactivated FVII content%,
The sum of the contents of the H chain and the L chain was defined as FVIIa content%. The activation rate was expressed as a percentage (%) obtained by dividing the FVIIa content by the sum of the FVII content and the FVIIa content.
【００１２】実施例１ 新鮮凍結血漿１００リットルを冷融解し沈澱画分を遠心分離
した上清を、陰イオン交換体(ＤＥＡＥ−セファデック
ス Ａ−５０:ファルマシア社)カラムに通液し、２０ｍ
Ｍクエン酸／０.１ＭＮａＣｌ緩衝液(ｐＨ７.０)にて充
分に洗浄し、２０ｍＭクエン酸／０.５ＭＮａＣｌ緩衝
液(ｐＨ７.０)にてＧｌａドメインを有するＰＰＳＢ画
分を溶出した。溶出液１０リットルを５０ｍＭＴｒｉｓ／１
５０ｍＭＮａＣｌ／５.０ｍＭＣａＣｌ2緩衝液(ｐＨ８.
０)で予め平衡化した抗ＦＶＩＩモノクローナル抗体固
定化アフィニティーゲルに展開し、５０ｍＭＴｒｉｓ／
２.５ＭＮａＣｌ／５.０ｍＭＣａＣｌ2緩衝液(ｐＨ８.
０)で洗浄後、５０ｍＭＴｒｉｓ／３０ｍＭＮａＣｌ／
５.０ｍＭＣａＣｌ2緩衝液(ｐＨ８.０)でさらに洗浄
し、５０ｍＭＴｒｉｓ／３０ｍＭＮａＣｌ／１０ｍＭＥ
ＤＴＡ緩衝液(ｐＨ７.４)で溶出してＦＶＩＩ画分を得
た。該ＦＶＩＩ画分を予め５０ｍＭＴｒｉｓ／３０ｍＭ
ＮａＣｌ緩衝液(ｐＨ８.０)で充填されているウイルス
除去膜(ベンベルグマイクロポーラスメンブレン、旭化
成)に展開し濾液を得た。得られた濾液のＦＶＩＩ純度
は８５％であった。 Example 1 100 liters of fresh frozen plasma was cooled and thawed, and the supernatant obtained by centrifuging the precipitated fraction was passed through an anion exchanger (DEAE-Sephadex A-50: Pharmacia) column to give 20 m
After washing thoroughly with M citric acid / 0.1 M NaCl buffer (pH 7.0), the PPSB fraction having a Gla domain was eluted with 20 mM citric acid / 0.5 M NaCl buffer (pH 7.0). Elute 10 liters of 50 mM Tris / 1
50 mM NaCl / 5.0 mM CaCl 2 buffer (pH 8.
The gel was developed on the affinity gel immobilized with the anti-FVII monoclonal antibody which had been equilibrated in
2.5 M NaCl / 5.0 mM CaCl 2 buffer (pH 8.
After washing with 0), 50 mM Tris / 30 mM NaCl /
Further washing with 5.0 mM CaCl 2 buffer (pH 8.0), 50 mM Tris / 30 mM NaCl / 10 mM E
Elution with DTA buffer (pH 7.4) gave the FVII fraction. The FVII fraction was previously purified to 50 mM Tris / 30 mM
The solution was developed on a virus removal membrane (Bemberg microporous membrane, Asahi Kasei) filled with a NaCl buffer (pH 8.0) to obtain a filtrate. The FVII purity of the obtained filtrate was 85%.
【００１３】上記０.２ｍｇ／ｍｌのＦＶＩＩ溶液５０
ｍｌを、予め５０ｍＭＴｒｉｓ／３０ｍＭＮａＣｌ緩衝
液(ｐＨ８.０)で平衡化した内径５.０ｍｍ、高さ５.０
ｃｍのＤＥＡＥ−セファロースファーストフロウ(ファ
ルマシア社)カラムに線速３００ｃｍ／ｈｒで展開し、
さらに同線速下、上記緩衝液で所定の各種カラム容量洗
浄し、吸着したＦＶＩＩ／ＦＶＩＩaを失活させるため
に５０ｍＭ酢酸緩衝液(ｐＨ３.５)で溶出した。溶出し
たＦＶＩＩ／ＦＶＩＩa画分のＳＤＳ−ＰＡＧＥ解析を
行ない、表１の結果を得た。本結果は洗浄カラム容量が
７０カラム容量を越えるとＦＶＩＩaの活性化率が６６.
４％を越え、ＧＤ-ｌｅｓｓＦＶＩＩaの産生が始まるこ
とを示している。The above-mentioned 0.2 mg / ml FVII solution 50
ml was previously equilibrated with a 50 mM Tris / 30 mM NaCl buffer (pH 8.0) and had an inner diameter of 5.0 mm and a height of 5.0.
cm DEAE-Sepharose Fast Flow (Pharmacia) column at a linear speed of 300 cm / hr,
Further, at the same linear velocity, the column was washed with the above-mentioned buffer at various column volumes, and eluted with a 50 mM acetate buffer (pH 3.5) to deactivate the adsorbed FVII / FVIIa. SDS-PAGE analysis of the eluted FVII / FVIIa fraction was performed, and the results in Table 1 were obtained. The results show that when the washing column volume exceeds 70 column volumes, the activation rate of FVIIa is 66.
It exceeds 4%, indicating that the production of GD-lessFVIIa starts.
【００１４】[0014]
【表１】 ＮＤは検出限界以下を意味する[Table 1] ND means below detection limit
【００１５】実施例２ 実施例１と同様な操作でウイルス除去膜濾液として調製
した０.２ｍｇ／ｍｌのＦＶＩＩ溶液５０ｍｌを、予め
５０ｍＭＴｒｉｓ／３０ｍＭＮａＣｌ緩衝液(ｐＨ８.
０)で平衡化した内径５.０ｍｍ、高さ５.０ｃｍのＤＥ
ＡＥ−セファロースファーストフロウ(ファルマシア社)
カラムに線速３００ｃｍ／ｈｒで展開し、さらに同線速
下、上記緩衝液で１０倍カラム容量洗浄し、５０ｍＭＴ
ｒｉｓ／３０ｍＭＮａＣｌ／２.０ｍＭＣａＣｌ2緩衝液
(ｐＨ８.０)を用い各種線速で溶出した。溶出したＦＶ
ＩＩ／ＦＶＩＩa画分のＳＤＳ−ＰＡＧＥ解析を行な
い、表２の結果を得た。本結果は溶出線速が１００ｃｍ
／ｈｒを下回るとＦＶＩＩaの活性化率が６２.８％を越
え、ＧＤ-ｌｅｓｓＦＶＩＩaの産生が始まることを示し
ている。 Example 2 50 ml of a 0.2 mg / ml FVII solution prepared as a virus removal membrane filtrate in the same manner as in Example 1 was previously charged with a 50 mM Tris / 30 mM NaCl buffer (pH 8.
DE with an inner diameter of 5.0 mm and a height of 5.0 cm equilibrated in 0)
AE-Sepharose First Flow (Pharmacia)
The column was developed at a linear speed of 300 cm / hr, and the column was washed 10 times with the above buffer at the same linear speed.
ris / 30 mM NaCl / 2.0 mM CaCl 2 buffer
(pH 8.0) and eluted at various linear velocities. Eluted FV
SDS-PAGE analysis of the II / FVIIa fraction was performed, and the results in Table 2 were obtained. This result shows that the elution linear velocity is 100 cm.
Below / hr, the activation rate of FVIIa exceeds 62.8%, indicating that the production of GD-lessFVIIa starts.
【００１６】[0016]
【表２】 ＮＤは検出限界以下を意味する[Table 2] ND means below detection limit
【００１７】実施例３ 実施例１と同様な操作でウイルス除去膜濾液として調製
した０.２ｍｇ／ｍｌのＦＶＩＩ溶液７０ｍｌを、予め
５０ｍＭＴｒｉｓ／３０ｍＭＮａＣｌ緩衝液(ｐＨ８.
０)で平衡化した内径５.０ｍｍ、高さ３.３ｃｍのＤＥ
ＡＥ−セファロースファーストフロウ(ファルマシア社)
カラムに線速２００ｃｍ／ｈｒで展開し、さらに同線速
下、上記緩衝液で１０カラム容量洗浄後、５０ｍＭＴｒ
ｉｓ／３０ｍＭＮａＣｌ／１.７５ｍＭＣａＣｌ2緩衝液
(ｐＨ８.０)を用い線速１５０ｃｍ／ｈｒで溶出した。
溶出した濃度２.０ｍｇ／ｍｌのＦＶＩＩ／ＦＶＩＩa混
合溶液をさらに液相中で１０時間熟成させた。カラム溶
出直後(熟成前)及び熟成後の成績を表３に示す。結果
は、実施例１、２とは陰イオン交換樹脂への展開量及び
カラムサイズの異なる条件においても、溶出後の活性化
率が６０％以下ではＧＤ-ｌｅｓｓＦＶＩＩaが産生され
ず、且つ熟成後もその産生が極めて抑制されていること
を示している。 Example 3 70 ml of a 0.2 mg / ml FVII solution prepared as a virus removal membrane filtrate by the same operation as in Example 1 was previously prepared in a 50 mM Tris / 30 mM NaCl buffer (pH 8.
DE with an inner diameter of 5.0 mm and a height of 3.3 cm equilibrated in 0)
AE-Sepharose First Flow (Pharmacia)
The column was developed at a linear speed of 200 cm / hr.
is / 30 mM NaCl / 1.75 mM CaCl 2 buffer
(pH 8.0) and eluted at a linear velocity of 150 cm / hr.
The eluted 2.0 mg / ml FVII / FVIIa mixed solution was further aged in the liquid phase for 10 hours. Table 3 shows the results immediately after column elution (before ripening) and after ripening. The results show that GD-lessFVIIa was not produced when the activation rate after elution was 60% or less even under the conditions different in the amount of development on the anion exchange resin and the column size from those in Examples 1 and 2, and even after aging. This indicates that its production is extremely suppressed.
【００１８】[0018]
【表３】 ＮＤは検出限界以下を意味する[Table 3] ND means below detection limit
【００１９】実施例４ 実施例１と同様な操作でウイルス除去膜濾液として調製
された０.２ｍｇ／ｍｌのＦＶＩＩ溶液３５ｍｌを予め
１０ｍＭＴｒｉｓ／１００ｍＭＮａＣｌ緩衝液(ｐＨ８.
５)で平衡化した内径５.０ｍｍ、高さ５.０ｃｍのＱ−
セファロースファーストフロウ(ファルマシア社)カラム
に線速３００ｃｍ／ｈｒで展開し、さらに同線速下、上
記緩衝液で各種カラム容量洗浄後、吸着したＦＶＩＩa
を失活させるために５０ｍＭ酢酸緩衝液(ｐＨ３.５)で
溶出した。溶出したＦＶＩＩ／ＦＶＩＩa画分のＳＤＳ
−ＰＡＧＥ解析を行ない、表４の結果を得た。本結果は
洗浄カラム容量が４０カラム容量を越えるとＦＶＩＩa
の活性化率が６１.３％を越え、ＧＤ-ｌｅｓｓＦＶＩＩ
aの産生が始まることを示している。 Example 4 35 ml of a 0.2 mg / ml FVII solution prepared as a virus removal membrane filtrate by the same operation as in Example 1 was previously prepared in a 10 mM Tris / 100 mM NaCl buffer (pH 8.
Q-equilibrated in 5) with an inner diameter of 5.0 mm and a height of 5.0 cm
FVIIa adsorbed on a Sepharose First Flow (Pharmacia) column at a linear speed of 300 cm / hr, washed at the same linear speed with various column volumes with the above buffer solution
Was eluted with 50 mM acetate buffer (pH 3.5) in order to inactivate. SDS of eluted FVII / FVIIa fraction
A PAGE analysis was performed, and the results in Table 4 were obtained. This result indicates that FVIIa was obtained when the washing column volume exceeded 40 column volumes.
Activation rate exceeds 61.3% and GD-lessFVII
This indicates that production of a begins.
【００２０】[0020]
【表４】 ＮＤは検出限界以下を意味する[Table 4] ND means below detection limit
【００２１】実施例５ 実施例１と同様な操作でウイルス除去膜濾液として調製
した０.２ｍｇ／ｍｌのＦＶＩＩ溶液３５ｍｌを、予め
１０ｍＭＴｒｉｓ／１００ｍＭＮａＣｌ緩衝液(ｐＨ８.
５)で平衡化した内径５.０ｍｍ、高さ５.０ｃｍのＱ−
セファロースファーストフロウ(ファルマシア社)カラム
に線速３００ｃｍ／ｈｒで展開し、さらに同線速下、上
記緩衝液で１０倍カラム容量洗浄し、１０ｍＭＴｒｉｓ
／１００ｍＭＮａＣｌ／４.０ｍＭＣａＣｌ2緩衝液(ｐ
Ｈ８.５)を用い各種線速で溶出した。溶出したＦＶＩＩ
／ＦＶＩＩa画分のＳＤＳ−ＰＡＧＥ解析を行ない、表
５の結果を得た。本結果は溶出線速が１０８ｃｍ／ｈｒ
以下になるとＦＶＩＩaの活性化率が６２.７％を越え、
ＧＤ-ｌｅｓｓＦＶＩＩaの産生が始まることを示してい
る。 Example 5 35 ml of a 0.2 mg / ml FVII solution prepared as a virus removal membrane filtrate by the same operation as in Example 1 was previously prepared in a 10 mM Tris / 100 mM NaCl buffer (pH 8.
Q-equilibrated in 5) with an inner diameter of 5.0 mm and a height of 5.0 cm
The column was developed on a Sepharose First Flow (Pharmacia) column at a linear speed of 300 cm / hr, and further washed with the above buffer at 10-fold column volume under the same linear speed.
/ 100 mM NaCl / 4.0 mM CaCl 2 buffer (p
H8.5) and eluted at various linear velocities. Eluted FVII
The SDS-PAGE analysis of the / FVIIa fraction was performed, and the results in Table 5 were obtained. This result shows that the elution linear velocity is 108 cm / hr.
When it becomes below, the activation rate of FVIIa exceeds 62.7%,
This indicates that the production of GD-lessFVIIa starts.
【００２２】[0022]
【表５】 ＮＤは検出限界以下を意味する[Table 5] ND means below detection limit
【００２３】実施例６ 実施例１と同様な操作でウイルス除去膜濾液として調製
した０.２ｍｇ／ｍｌのＦＶＩＩ溶液３５ｍｌを、予め
１０ｍＭＴｒｉｓ／１００ｍＭＮａＣｌ緩衝液(ｐＨ８.
５)で平衡化した内径５.０ｍｍ、高さ５.０ｃｍのＱ−
セファロースファーストフロウ(ファルマシア社)カラム
に線速３００ｃｍ／ｈｒで展開し、さらに同線速下、上
記緩衝液で１０カラム容量洗浄後、１０ｍＭＴｒｉｓ／
１００ｍＭＮａＣｌ／４.０ｍＭＣａＣｌ2緩衝液(ｐＨ
８.５)で線速１８０ｃｍ／ｈｒで溶出した。溶出した濃
度１.０ｍｇ／ｍｌのＦＶＩＩ／ＦＶＩＩa混合溶液を液
相中で２５時間熟成させた。カラム溶出直後(熟成前)及
び熟成後の成績を表６に示す。結果は、実施例３と同様
に陰イオン交換樹脂としてＱ−セファロースファースト
フロウ(ファルマシア社)を使用しても、溶出後の活性化
率が６０％以下ではＧＤ-ｌｅｓｓＦＶＩＩaが産生され
ず、且つ溶出液熟成後もその産生が極めて抑制されてい
ることを示している。 Example 6 35 ml of a 0.2 mg / ml FVII solution prepared as a virus removal membrane filtrate by the same operation as in Example 1 was previously prepared in a 10 mM Tris / 100 mM NaCl buffer (pH 8.
Q-equilibrated in 5) with an inner diameter of 5.0 mm and a height of 5.0 cm
The column was developed on a Sepharose Fast Flow (Pharmacia) column at a linear speed of 300 cm / hr, and at the same linear speed, 10 column volumes were washed with the above buffer solution, and then 10 mM Tris /
100 mM NaCl / 4.0 mM CaCl 2 buffer (pH
Eluted at 8.5) at a linear velocity of 180 cm / hr. The eluted FVII / FVIIa mixed solution having a concentration of 1.0 mg / ml was aged in the liquid phase for 25 hours. Table 6 shows the results immediately after elution of the column (before ripening) and after ripening. As a result, even when Q-Sepharose Fast Flow (Pharmacia) was used as the anion exchange resin as in Example 3, GD-lessFVIIa was not produced when the activation rate after elution was 60% or less, and the elution was not achieved. This indicates that the production is extremely suppressed even after liquid ripening.
【００２４】[0024]
【表６】 ＮＤは検出限界以下を意味する[Table 6] ND means below detection limit
【００２５】実施例７ 実施例３と同様な操作で、ＤＥＡＥ−セファロースファ
ーストフロウ(ファルマシア社)を使用し部分活性化後、
液相で熟成し完全活性化させたＣａ2+濃度２.０ｍＭを
含む５.０ｍｇ／ｍｌ濃度のＦＶＩＩa混合溶液を、Ｃａ
2+濃度が０.１７ｍＭ、０.５０ｍＭ、０.７５ｍＭ、１.
０ｍＭ、２.０ｍＭ、蛋白質濃度が０.４ｍｇ／ｍｌにな
るように５０ｍＭＴｒｉｓ／３０ｍＭＮａＣｌ緩衝液
(ｐＨ８.１)で希釈し、９℃で８８時間インキュベート
した。インキュベート後の成績を表７に記載する。表７
の成績はＧＤ-ｌｅｓｓＦＶＩＩa産生の至適Ｃａ2+濃度
が０.５ｍＭ未満であることを示すと共に、ＦＶＩＩaの
ＧＤ-ｌｅｓｓＦＶＩＩaへの分解反応が０.７５ｍＭ以
上のＣａ2+濃度下で抑制されることをも示している。こ
の結果は液相におけるＧＤ-ｌｅｓｓＦＶＩＩa産生反応
のＣａ2+濃度の閾値を示すと共に、陰イオン交換樹脂か
らの溶出をＣａ2+を含む緩衝液で行なう場合、溶出時の
ＧＤ-ｌｅｓｓＦＶＩＩaの産生を抑制するためには溶出
緩衝液のＣａ2+濃度は０.５ｍＭ以上必要であることを
示している。 Example 7 In the same manner as in Example 3, after partially activating using DEAE-Sepharose Fast Flow (Pharmacia),
A 5.0 mg / ml FVIIa mixed solution containing 2.0 mM Ca 2+ , which was aged and completely activated in the liquid phase, was mixed with Ca
2+ concentrations of 0.17 mM, 0.50 mM, 0.75 mM, 1.
0 mM, 2.0 mM, 50 mM Tris / 30 mM NaCl buffer so that the protein concentration becomes 0.4 mg / ml.
(pH 8.1) and incubated at 9 ° C. for 88 hours. The results after the incubation are described in Table 7. Table 7
Shows that the optimal Ca 2+ concentration for GD-lessFVIIa production is less than 0.5 mM, and that the decomposition reaction of FVIIa to GD-lessFVIIa is suppressed at a Ca 2+ concentration of 0.75 mM or more. It also shows that. This result shows the threshold value of the Ca 2+ concentration in the GD-less FVIIa production reaction in the liquid phase, and when the elution from the anion exchange resin is performed using a buffer containing Ca 2+ , the production of GD-less FVIIa during the elution is reduced. This indicates that the concentration of Ca 2+ in the elution buffer must be 0.5 mM or more for suppression.
【００２６】[0026]
【表７】 [Table 7]
───────────────────────────────────────────────────── フロントページの続き (72)発明者 丸野 真一 熊本県菊池郡西合志町須屋1733−31 (72)発明者 緒方 洋一 熊本県熊本市帯山７丁目９−142 (72)発明者 寺野 剛 熊本県菊池郡西合志町須屋1548−２ ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Shinichi Maruno 1733-31, Suya, Nishigoshi-cho, Kikuchi-gun, Kumamoto Prefecture (72) Inventor Yoichi Ogata 7-9-142 Obiyama, Kumamoto City, Kumamoto Prefecture 1548-2 Suya, Nishikoshi-cho, Kikuchi-gun
Claims (6)
Ｉ因子(以下、ＦＶＩＩと称することがある)の活性化血
液凝固第ＶＩＩ因子(以下、ＦＶＩＩaと称することがあ
る)への活性化方法。ＦＶＩＩを含有する溶液を陰イ
オン交換樹脂に接触させ展開し、ＦＶＩＩを部分的に
活性化し溶出して、該溶出液を溶液状態で静置し未活
性体分子を完全に活性化させる。1. A blood coagulation VI comprising the following steps:
A method for activating factor I (hereinafter sometimes referred to as FVII) to activated blood coagulation factor VII (hereinafter sometimes referred to as FVIIa). The solution containing FVII is brought into contact with the anion exchange resin and developed, FVII is partially activated and eluted, and the eluate is left in a solution state to completely activate the inactive molecule.
率が６０％未満である請求項１記載のＦＶＩＩの活性化
方法。2. The method for activating FVII according to claim 1, wherein the activation rate of the eluate after the partial activation is less than 60%.
溶出緩衝液はＣａ2 +を含有し該Ｃａ2+濃度が０.５ｍＭ
以上である請求項１または請求項２記載のＦＶＩＩの活
性化方法。3. The elution buffer for elution from the anion exchange resin contains Ca 2 + , and the Ca 2+ concentration is 0.5 mM.
The method for activating FVII according to claim 1 or 2, which is described above.
溶出方法が段階的溶出法(ステップワイズ溶出法)である
請求項１から請求項３のいずれかに記載のＦＶＩＩの活
性化方法。4. The method for activating FVII according to claim 1, wherein the elution method at the time of elution from the anion exchange resin is a stepwise elution method (stepwise elution method).
溶液中にＣａ2+を含有し該Ｃａ2+濃度が０.５ｍＭ以上
である請求項１に記載のＦＶＩＩの活性化方法。5. The method for activating FVII according to claim 1, wherein the solution contains Ca 2+ in the solution when left standing (aging) in a solution state, and the Ca 2+ concentration is 0.5 mM or more.
載のＦＶＩＩの活性化方法に基づく工程を含むことを特
徴とする活性化血液凝固第ＶＩＩ因子の製造方法。6. A method for producing activated blood coagulation factor VII, comprising a step based on the method for activating FVII according to any one of claims 1 to 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23714596A JP4046377B2 (en) | 1996-08-19 | 1996-08-19 | Method for activating blood coagulation factor VII and method for producing activated blood coagulation factor VII based on the method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23714596A JP4046377B2 (en) | 1996-08-19 | 1996-08-19 | Method for activating blood coagulation factor VII and method for producing activated blood coagulation factor VII based on the method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH1059867A true JPH1059867A (en) | 1998-03-03 |
| JP4046377B2 JP4046377B2 (en) | 2008-02-13 |
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ID=17011081
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23714596A Expired - Lifetime JP4046377B2 (en) | 1996-08-19 | 1996-08-19 | Method for activating blood coagulation factor VII and method for producing activated blood coagulation factor VII based on the method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000055203A1 (en) * | 1999-03-15 | 2000-09-21 | Novo Nordisk A/S | Ion exchange chromatographic separation of glp-1 and related peptides |
| US6881721B2 (en) | 1999-12-24 | 2005-04-19 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Medicinal compositions for treating and preventing diseases based on abnormal blood coagulation |
| US7749955B2 (en) | 2003-08-21 | 2010-07-06 | Novo Nordisk A/S | Separation of polypeptides comprising a racemized amino acid |
| US9488625B2 (en) | 2010-12-15 | 2016-11-08 | Baxalta GmbH | Purification of factor VIII using a conductivity gradient |
-
1996
- 1996-08-19 JP JP23714596A patent/JP4046377B2/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000055203A1 (en) * | 1999-03-15 | 2000-09-21 | Novo Nordisk A/S | Ion exchange chromatographic separation of glp-1 and related peptides |
| US6881721B2 (en) | 1999-12-24 | 2005-04-19 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Medicinal compositions for treating and preventing diseases based on abnormal blood coagulation |
| US7749955B2 (en) | 2003-08-21 | 2010-07-06 | Novo Nordisk A/S | Separation of polypeptides comprising a racemized amino acid |
| US9488625B2 (en) | 2010-12-15 | 2016-11-08 | Baxalta GmbH | Purification of factor VIII using a conductivity gradient |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4046377B2 (en) | 2008-02-13 |
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