JP4855074B2 - High-purity blood coagulation factor IX preparation and purification method thereof - Google Patents

High-purity blood coagulation factor IX preparation and purification method thereof Download PDF

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JP4855074B2
JP4855074B2 JP2005514403A JP2005514403A JP4855074B2 JP 4855074 B2 JP4855074 B2 JP 4855074B2 JP 2005514403 A JP2005514403 A JP 2005514403A JP 2005514403 A JP2005514403 A JP 2005514403A JP 4855074 B2 JP4855074 B2 JP 4855074B2
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純一 下田
寿 矢野
美奈子 宮川
まい 東
洋一 緒方
高義 濱本
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Description

本発明は、血液凝固IX因子(以下、FIX)の精製方法および当該精製方法によって得られたより高純度のFIX調製物に関する。詳細には、FIX含有画分からFIX活性化因子、特にマンノース結合レクチン結合性セリンプロテアーゼ−1(MASP−1)を除去することによるFIXの精製方法、および当該精製方法を経て得られるより高純度のFIX精製画分およびFIX製剤に関する。  The present invention relates to a method for purifying blood coagulation factor IX (hereinafter referred to as FIX) and a higher-purity FIX preparation obtained by the purification method. Specifically, a FIX purification method by removing a FIX activator, particularly mannose-binding lectin-binding serine protease-1 (MASP-1), from the FIX-containing fraction, and a higher-purity obtained through the purification method The present invention relates to a FIX purified fraction and a FIX preparation.

FIXはセリンプロテアーゼの前駆体であり、アミノ末端領域にγ−カルボキシグルタミン酸(Gla)残基を有するビタミンK依存性血液凝固因子である。FIXは肝臓で合成され、血中には3〜5μg/ml存在する。FIXは、生理的には活性化血液凝固VII因子(以下、FVIIa)−組織因子(TF)複合体、あるいは活性化血液凝固XI因子(以下、FXIa)により活性化されることが知られている。活性化により生成した活性化血液凝固IX因子(以下、FIXa)は、さらに血液凝固X因子(以下、FX)を活性化し血液凝固を進行させる。ヒトFIXは分子量57,000の一本鎖糖タンパク質で、アミノ末端からGlaドメイン、ヒンジ領域、2つのEGF様ドメイン、さらに活性化ペプチド領域を経てセリンプロテアーゼドメインというドメイン配列により構成されている。Glaドメインには12個のGla残基が存在している。  FIX is a precursor of serine protease and is a vitamin K-dependent blood coagulation factor having a γ-carboxyglutamic acid (Gla) residue in the amino terminal region. FIX is synthesized in the liver and is present in the blood at 3-5 μg / ml. It is known that FIX is physiologically activated by activated blood coagulation factor VII (hereinafter referred to as FVIIa) -tissue factor (TF) complex, or activated blood coagulation factor XI (hereinafter referred to as FXIa). . The activated blood coagulation factor IX (hereinafter referred to as FIXa) generated by the activation further activates blood coagulation factor X (hereinafter referred to as FX) to advance blood coagulation. Human FIX is a single-chain glycoprotein with a molecular weight of 57,000, and is composed of a domain sequence called a serine protease domain from the amino terminus via a Gla domain, a hinge region, two EGF-like domains, and an activated peptide region. There are 12 Gla residues in the Gla domain.

FIXの欠乏/欠損に基づく異常症は、出血性疾患である血友病Bとして知られている。血友病Bに対する根治的療法は現在のところなく、FIXの補充療法による患者の止血管理が治療の主体である。  An abnormality based on FIX deficiency / deficiency is known as hemophilia B, a bleeding disorder. There is no curative therapy for hemophilia B at present, and the management of hemostasis in patients with FIX replacement therapy is the main treatment.

1980年代には、クリオ上清やコーンのエタノール分画のフラクションI上清などを出発原料として、陰イオン交換体を用いたクロマトグラフィーで分画して精製される血液凝固IX因子複合体製剤(PCC製剤)がFIXの補充療法に使用されていた(例えば、非特許文献1、非特許文献2、および非特許文献3参照)。また、FIXは、その他のビタミンK依存性の凝固因子(プロトロンビン、FVII、FXなど)と物理化学的に性状が類似しているために、それらとの分離が困難であったが、アフィニティークロマトグラフィー精製技術の発達により、高純度FIXの精製が可能になった。例えば、1980年代後半には、ヘパリンをリガンドにした担体を用いたアフィニティークロマトグラフィーによりFIXを高度に精製したFIX製剤が臨床使用されるようになった(例えば、非特許文献4、非特許文献5、および非特許文献6参照)。さらに1990年代になると、FIXに対するモノクローナル抗体を用いたイムノアフィニティークロマトグラフィーでFIX以外のビタミンK依存性の凝固因子を除去し、FIXを特異的に高度に精製したFIX製剤が使用されるようになってきた(例えば、非特許文献7、非特許文献8、および非特許文献9参照)。  In the 1980s, a blood coagulation factor IX complex preparation prepared by fractionating and purifying by chromatography using an anion exchanger using a cryo-supernatant or a fraction I supernatant of ethanol fraction of corn as a starting material ( PCC preparations) have been used for FIX replacement therapy (see, for example, Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3). FIX is difficult to separate from other vitamin K-dependent coagulation factors (prothrombin, FVII, FX, etc.) because of its physicochemical properties, but affinity chromatography. With the development of purification technology, high purity FIX can be purified. For example, in the latter half of the 1980s, FIX preparations obtained by highly purifying FIX by affinity chromatography using a carrier having heparin as a ligand were clinically used (for example, Non-Patent Document 4 and Non-Patent Document 5). And Non-Patent Document 6). Further, in the 1990s, FIX preparations in which FIX was highly purified specifically were obtained by removing vitamin K-dependent coagulation factors other than FIX by immunoaffinity chromatography using a monoclonal antibody against FIX. (For example, see Non-Patent Document 7, Non-Patent Document 8, and Non-Patent Document 9).

Michalski C.ら,Vox Sanguinis,55巻,4号,1988年,p.202−210Michaelski C.I. Vox Sanguinis, 55, No. 4, 1988, p. 202-210 神谷忠,日本輸血学会誌,33巻,5号,1987年,p.638−644Kamiya Tadashi, Journal of Japanese Society of Blood Transfusion, Vol. 33, No. 5, 1987, p. 638-644 安田純一,血液製剤,近代出版,1986年,p.184−191Junichi Yasuda, Blood Products, Modern Publishing, 1986, p. 184-191 Burnouf T.ら,Journal of Biochemical and Biophysical Methods,49巻,1−3号,2001年,p.575−586Burnow T. Et al., Journal of Biochemical and Biophysical Methods, 49, 1-3, 2001, p. 575-586 Josic D.ら,Journal of Chromatography,632巻,1−2号,1993年,p.1−10Josic D. Et al., Journal of Chromatography, 632, 1-2, 1993, p. 1-10 Burnouf T.ら,Vox Sanguinis,57巻,4号,1989年,p.225−232Burnow T. Et al., Vox Sanguinis, 57, 4, 1989, p. 225-232 森河亘ら,基礎と臨床,24巻,10号,1990年,713−717頁Wataru Morikawa et al., Fundamental and Clinical, 24, 10, 1990, 713-717 Iga Y.ら,Proceedings of the Sixth International Symposium on Hemophilia Treatment,Tokyo,Japan,1990年,p.107−116Iga Y. Et al., Proceedings of the Sixth International Symposium on Hemophilia Treatment, Tokyo, Japan, 1990, p. 107-116 Akimoto Y.ら,Proceedings of the sixth International Symposium on Hemophilia Treatment,Tokyo,Japan,1990年,p.117−130Akimoto Y. et al. Et al., Proceedings of the Sixth International Symposium on Hemophilia Treatment, Tokyo, Japan, 1990, p. 117-130

しかし、アフィニティークロマトグラフィーあるいはイムノアフィニティークロマトグラフィーで高純度に精製されたFIX製剤であっても、精製工程中に生成した微量のFIXa(FIX量に対して0.1〜1.0%)が含まれる。FIXaとウサギ鬱血試験(血栓傾向を検査する生体内試験)のスコアーとの間に正相関があるとの報告もある(例えば、Gray E.ら,Thrombosis and Haemostasis,73巻,4号,1995年,p.675−679およびPhilippou H.ら,Thrombosis and Haemostasis,76巻,1号,1996年,p.23−28参照)。したがって、高純度に精製されたFIX調製物中になお微量に混在するFIXaを可能な限り低減させることは、血栓誘発の危険性をさらに低減させ、FIX製剤の安全性をより高めるものである。  However, even a FIX preparation purified to high purity by affinity chromatography or immunoaffinity chromatography contains a small amount of FIXa (0.1 to 1.0% based on the amount of FIX) generated during the purification process. It is. There is also a report that there is a positive correlation between the score of FIXa and the rabbit stasis test (in vivo test to examine thrombus tendency) (for example, Gray E. et al., Thrombosis and Haemostasis, Vol. 73, No. 4, 1995). , P.675-679 and Philippou H. et al., Thrombosis and Haemostasis, 76, No. 1, 1996, p.23-28). Therefore, reducing FIXa that is still mixed in a trace amount in a highly purified FIX preparation as much as possible further reduces the risk of thrombus induction and further increases the safety of the FIX preparation.

ここで、FIX製剤中のFIXaを低減させる方策としては、一旦生成したFIXaをFIX製剤の最終組成物となる以前の精製工程で除去することの他に、FIXa生成の原因となるFIX活性化因子をFIX製造工程中で予め除き、精製されたFIX調製物中にFIXaが混入されないようにするという対応策も考えられた。  Here, as a measure for reducing FIXa in the FIX preparation, in addition to removing FIXa once produced in the purification step before becoming the final composition of the FIX preparation, a FIX activator causing FIXa generation A countermeasure was also conceived in which the FIXa was removed in advance during the FIX manufacturing process so that FIXa was not mixed into the purified FIX preparation.

本発明者らは、ヒト血漿から陰イオン交換体を用いる既報告の方法(例えば、森河亘ら,基礎と臨床,24巻,10号,1990年,713−717頁参照)で精製分離したビタミンK依存性凝固因子画分を出発原料として、FIXに対するモノクローナル抗体を用いたイムノアフィニティークロマトグラフィーで精製したFIXサンプル(M画分)の溶液をインキュベーションするとFIXが活性化し、FIXaに変換されることを見出していた。このFIXaの生成速度は、陰イオン交換体ゲルと混合すると増加した。  The present inventors have purified and separated vitamins from human plasma by a reported method using an anion exchanger (see, for example, Wataru Morikawa et al., Basic and Clinical, 24, 10, 1990, pages 713-717). Incubation of a solution of FIX sample (M fraction) purified by immunoaffinity chromatography using a monoclonal antibody against FIX using the K-dependent coagulation factor fraction as a starting material indicates that FIX is activated and converted to FIXa. I was heading. The rate of FIXa formation increased when mixed with an anion exchanger gel.

この原因は、FIXの生理的な活性化因子であるといわれているFXIaやFVIIaが微量に混在しているためであろうと当初は推測された。しかし、M画分サンプル中のFXIaとFVIIa含量をELISAにて調べ、あらかじめ高純度に精製したFIXにM画分と同等量のFXIaあるいはFVIIaを添加し反応させ、それらによるFIX活性化能を調べた結果、FXIaあるいはFVIIaを添加したサンプルよりも、M画分サンプルのFIX活性化能が著しく高いことが示された。この事実より、FXIaおよびFVIIa以外の新規なFIX活性化プロテアーゼの存在が示唆された。  It was initially speculated that this was probably due to a small amount of FXIa or FVIIa, which is said to be a physiological activator of FIX. However, the contents of FXIa and FVIIa in the M fraction sample were examined by ELISA, and FXIa or FVIIa in the same amount as the M fraction was added to the FIX purified in advance and reacted, and the FIX activation ability by them was examined. As a result, it was shown that the FIX activation ability of the M fraction sample was significantly higher than that of the sample to which FXIa or FVIIa was added. This fact suggested the existence of novel FIX activating proteases other than FXIa and FVIIa.

一方、レクチン経路と呼ばれる第3の補体活性化経路で働くセリンプロテアーゼの一種にマンノース結合レクチン結合性セリンプロテアーゼ(MASP)がある(例えば、Sato T.ら,International Immunology,6巻,4号,1994年,p.665−669;Endo Y.ら,Journal of Immunology,161巻,9号,1998年,p.4924−4930;および遠藤雄一ら,蛋白質 核酸 酵素,45巻,5号,2000年,p.671−678参照)。このMASPには、MASP−1、MASP−2、MASP−3、およびsMAPの4タイプがあることが知られている。しかしながら、MASPが、血液凝固因子の活性化に関与することは従来知られていなかった。  On the other hand, mannose-binding lectin-binding serine protease (MASP) is one type of serine protease that works in the third complement activation pathway called lectin pathway (for example, Sato T. et al., International Immunology, Vol. 6, No. 4, 1994, p.665-669; Endo Y. et al., Journal of Immunology, 161, 9, 1998, p.4924-4930; and Yuichi Endo, et al., Protein Nucleic Acid Enzyme, 45, 5, 2000. , P.671-678). It is known that there are four types of MASPs: MASP-1, MASP-2, MASP-3, and sMAP. However, it has not been conventionally known that MASP is involved in the activation of blood coagulation factors.

本発明者らは、上記の諸問題に鑑み鋭意検討した結果、精製MASP−1が時間依存的にFIXを活性化すること、およびヒトMASP−1に対するモノクローナル抗体を固定化したゲルを用いてM画分からMASP−1を除去すると、FIX活性化の程度が激減(約95%の活性化能が消失)することを見出し、これら事実からFIX活性化プロテアーゼの主体がMASPの一種であるMASP−1であることを確認した。このようにして、アフィニティークロマトグラフィーあるいはイムノアフィニティークロマトグラフィーで高純度に精製された従来のFIX製剤中になお微量のFIXa(FIX量に対して0.1〜1.0%)が含まれる原因は、MASP−1の混入による精製工程中でのFIXの活性化にあることが判明した。  As a result of intensive studies in view of the above-mentioned problems, the present inventors have found that purified MASP-1 activates FIX in a time-dependent manner, and a gel on which a monoclonal antibody against human MASP-1 is immobilized. When MASP-1 was removed from the fraction, it was found that the degree of FIX activation drastically decreased (approximately 95% of the activation ability disappeared). From these facts, MASP-1 whose main component of FIX activating protease is a kind of MASP is found. It was confirmed that. In this way, the reason why a small amount of FIXa (0.1 to 1.0% relative to the amount of FIX) is still contained in the conventional FIX preparation purified to high purity by affinity chromatography or immunoaffinity chromatography is as follows. It was found that FIX was activated during the purification process due to contamination with MASP-1.

すなわち、本発明は、高純度に精製された従来のFIX製剤中になお微量に混在しているFIXaの原因が、これまでFIXの生理的な活性化因子であることが知られているFXIaやFVIIaとは異なる新規なFIX活性化因子であるMASP、特にMASP−1によるものであるとの上記知見に基づき、FIX含有画分から当該FIX活性化因子(MASP、特にMASP−1)を除去することを特徴とするFIXの精製方法に関するものである。本発明により、高純度に精製されたFIX調製物中になお微量に混在するFIXaが可能な限り低減された、さらに安全性の高いFIX製剤を提供することが可能となる。  That is, the present invention relates to FXIa, which has been known to cause FIX's physiological activator until now because the cause of FIXa that is still mixed in a trace amount in a conventional FIX preparation purified to a high purity. Based on the above finding that MASP, in particular MASP-1, is a novel FIX activator different from FVIIa, the FIX activator (MASP, especially MASP-1) is removed from the FIX-containing fraction It is related with the purification method of FIX characterized by these. According to the present invention, it is possible to provide a highly safe FIX preparation in which the amount of FIXa mixed in a trace amount in a highly purified FIX preparation is reduced as much as possible.

また、本発明は、本発明による精製方法によって得られた、FIX1国際単位当たりのMASP−1含有量が0.1ng以下のFIX精製画分に関する。  The present invention also relates to a FIX purified fraction having a MASP-1 content of 0.1 ng or less per FIX1 international unit obtained by the purification method of the present invention.

また、本発明は、FIXに対するFIXaの含有率が0.1%以下のFIX製剤に関する。当該FIX製剤は、さらにFIX1国際単位当たりのMASP−1含有量が0.1ng以下である。  The present invention also relates to a FIX preparation having a FIXa content of 0.1% or less with respect to FIX. The FIX preparation further has a MASP-1 content of 0.1 ng or less per FIX1 international unit.

本発明により、血友病B患者に対する補充療法剤として汎用されているFIX製剤に関して、微量に存在するFIXaが従来の製剤よりも有意に低減し、血栓誘発の危険性の少ないより安全性の高い製剤を提供することが可能となった。  According to the present invention, FIXa present in a trace amount is significantly reduced as compared with conventional preparations and is more safe with less risk of thrombus induction, with respect to FIX preparations widely used as replacement therapy agents for hemophilia B patients. It became possible to provide a formulation.

当該FIX活性化因子の除去方法としては、「MASP−1に対するモノクローナル抗体を固定化したゲルを用いてFIX画分からMASP−1を除去する方法」、「セファロース担体のカラムを通すことでFIX画分からMASP−1を吸着除去する方法」、および「陰イオン交換ゲル、疎水ゲル、キレートゲル、ベンズアミジン固定化ゲルを用いたクロマトグラフィーによりFIX画分からMASP−1を除去する方法」などがあるが、疎水クロマトグラフィーを実施する方法は特に好ましい態様である。  As a method for removing the FIX activator, “a method for removing MASP-1 from a FIX fraction using a gel in which a monoclonal antibody against MASP-1 is immobilized”, “from a FIX fraction by passing through a Sepharose carrier column”. “Method for removing MASP-1 by adsorption” and “Method for removing MASP-1 from FIX fraction by chromatography using anion exchange gel, hydrophobic gel, chelate gel, benzamidine-immobilized gel”, etc. The method of performing chromatography is a particularly preferred embodiment.

本発明によりMASP−1を除去するには疎水クロマトグラフィーを利用するのが好ましい。その場合、イムノアフィニティーあるいはヘパリン様物質をリガンドとした担体で精製した高純度FIXの高食塩濃度溶液を疎水担体に展開してFIXを吸着させ、MASP−1を素通りさせる。FIXは低食塩濃度の緩衝液で特異的に溶出される。吸着の際の食塩濃度は1M以上が好ましく、溶出時は1M以下が好ましい。  Hydrophobic chromatography is preferably used to remove MASP-1 according to the present invention. In that case, a high salt solution of high purity FIX purified with a carrier having immunoaffinity or heparin-like substance as a ligand is developed on a hydrophobic carrier to adsorb FIX, and MASP-1 is passed through. FIX is specifically eluted with low saline buffer. The salt concentration at the time of adsorption is preferably 1M or more, and preferably 1M or less at the time of elution.

本発明の疎水クロマトグラフィーに用いる担体のリガンドは、ブチル基、フェニル基あるいはオクチル基が好ましく、担体はトヨパール(製品名:東ソー(株))をはじめ種々のものを選択可能である。  The ligand of the carrier used in the hydrophobic chromatography of the present invention is preferably a butyl group, a phenyl group or an octyl group, and various carriers such as Toyopearl (product name: Tosoh Corporation) can be selected.

また、本発明の疎水クロマトグラフィーでの温度は1℃〜30℃が好ましく、pHは5〜8が好ましい。  Further, the temperature in the hydrophobic chromatography of the present invention is preferably from 1 ° C to 30 ° C, and the pH is preferably from 5 to 8.

このようにしてFIX精製画分に混入したMASP−1を有効に除去することで、FIX精製画分に微量に混在するFIXaを可能な限り低減させ、血栓誘発の危険性をさらに低下した安全性の高いFIX製剤を提供することができる。  By effectively removing MASP-1 mixed in the FIX purified fraction in this way, FIXa mixed in a trace amount in the FIX purified fraction is reduced as much as possible, and the risk of further thrombus induction is further reduced. High FIX formulation can be provided.

以下、本発明の実施例を血液凝固IX因子(FIX)の調製例とともに示し、本発明をさらに具体的に説明するが、本発明はこれに限定されるものではない。  Examples of the present invention are shown below together with preparation examples of blood coagulation factor IX (FIX), and the present invention will be described more specifically. However, the present invention is not limited thereto.

《1.血液凝固IX因子(FIX)の調製》
ヒト血漿3000mlからクリオ沈殿を除去したクリオ上清を、20mMクエン酸緩衝液で平衡化した陰イオン交換体(Q−Sepharose)100mlに通液し、吸着画分を0.5Mの食塩を含むクエン酸緩衝液で溶出し、FIX含有画分を得た。
<< 1. Preparation of blood coagulation factor IX (FIX) >>
The cryo supernatant after removing cryoprecipitate from 3000 ml of human plasma is passed through 100 ml of an anion exchanger (Q-Sepharose) equilibrated with 20 mM citrate buffer, and the adsorbed fraction is quenched with 0.5 M sodium chloride. Elution with an acid buffer gave a FIX-containing fraction.

得られたFIX画分に、終濃度50mMになるようにカルシウムを添加し、カルシウム依存性の抗FIXモノクローナル抗体をリガンドとした担体(100ml)に通液しFIXを担体に吸着させた。担体を洗浄し、20mMのEDTA含有緩衝液でFIXを溶出し、高純度化したFIXを得た。  Calcium was added to the obtained FIX fraction so as to have a final concentration of 50 mM, and the solution was passed through a carrier (100 ml) having a calcium-dependent anti-FIX monoclonal antibody as a ligand to adsorb FIX on the carrier. The carrier was washed, and FIX was eluted with a 20 mM EDTA-containing buffer to obtain highly purified FIX.

《2.疎水クロマトグラフィーによるMASP−1の除去およびFIXの調製》
上記により調製されたFIX溶液に終濃度1.5Mになるように食塩を添加し、クロマトグラフィーアプライサンプルを調製した。次に、25℃にて、そのサンプルを、ヒスチジン緩衝液(pH6)で平衡化したブチルトヨパール650C(製品名:東ソー(株))20mlに通液しFIXを吸着させた。1.1M食塩含有緩衝液で洗浄後、0.5M食塩含有緩衝液でFIXを溶出した。疎水クロマトグラフィー精製後のFIXは、森河らの方法(森河亘ら,基礎と臨床,24巻,10号,1990年,713−717頁)に準じて、Q−Sepharoseによりさらに高純度に精製された。得られたFIX画分におけるMASP−1含量およびFIXa含量を、疎水クロマトグラフィーを実施しない従来法により得られるFIX画分の成績と比較したものを、表1に示した。疎水クロマトグラフィーを導入することにより、FIX1国際単位当たりのMASP−1含量は0.1ng以下まで低下し、それに伴いFIXに対するFIXaの含有率も0.1%以下まで低下した。
<< 2. Removal of MASP-1 and Preparation of FIX by Hydrophobic Chromatography >>
Sodium chloride was added to the FIX solution prepared above to a final concentration of 1.5 M to prepare a chromatographic apply sample. Next, at 25 ° C., the sample was passed through 20 ml of butyltoyopearl 650C (product name: Tosoh Corporation) equilibrated with histidine buffer (pH 6) to adsorb FIX. After washing with 1.1 M sodium chloride-containing buffer, FIX was eluted with 0.5 M sodium chloride-containing buffer. The FIX after purification by hydrophobic chromatography is further purified with Q-Sepharose according to the method of Morikawa et al. (Morikawa Wata et al., Basic and Clinical, 24, 10, 1990, 713-717). It was. Table 1 shows a comparison of the MASP-1 content and the FIXa content in the obtained FIX fraction with the results of the FIX fraction obtained by the conventional method without performing hydrophobic chromatography. By introducing hydrophobic chromatography, the MASP-1 content per FIX1 international unit decreased to 0.1 ng or less, and accordingly, the content of FIXa relative to FIX also decreased to 0.1% or less.

Figure 0004855074
Figure 0004855074

Claims (3)

血液凝固IX因子の精製方法であって、血液凝固IX因子に対する抗体を用いたイムノアフィニティークロマトグラフィー処理により得られる、血漿由来の高純度血液凝固IX因子含有画分を出発材料とし、さらに疎水クロマトグラフィー処理することを含み、該疎水クロマトグラフィー処理は、1Mよりも高く1.5M以下のNaCl濃度およびpH6以上でpH7よりも低いpHで血液凝固IX因子をクロマトグラフィー担体に吸着させ、1Mよりも高く1.1M以下のNaCl濃度およびpH6以上でpH7よりも低いpHで担体を洗浄してマンノース結合レクチン結合性セリンプロテアーゼ−1(以下、MASP-1)を素通りさせ、除去するものであり、血液凝固IX因子1国際単位当たりのMASP-1の含有量が0.1ng以下であることを特徴とする方法 A method for purifying blood coagulation factor IX, which is obtained by subjecting a plasma-derived fraction containing high-purity blood coagulation factor IX obtained by immunoaffinity chromatography using an antibody against blood coagulation factor IX to hydrophobic chromatography. The hydrophobic chromatographic treatment comprises adsorbing blood coagulation factor IX to the chromatographic support at a NaCl concentration greater than 1M and less than 1.5M and at a pH greater than 6 and less than pH 7, and greater than 1M. 1. Washing the carrier at a NaCl concentration of 1.1 M or less and a pH of 6 or higher and lower than pH 7 to allow mannose-binding lectin-binding serine protease-1 (hereinafter referred to as MASP-1) to pass through and remove the blood coagulation and the content of MASP-1 per factor IX 1 international unit is less than 0.1 ng, method. 血液凝固IX因子に対する活性化血液凝固IX因子の含有率が0.1%以下であり、血液凝固IX因子1国際単位当たりのMASP-1の含有量が0.1ng以下であることを特徴とする、血漿由来の血液凝固IX因子製剤。The content of activated blood coagulation factor IX relative to blood coagulation factor IX is 0.1% or less, and the content of MASP-1 per international unit of blood coagulation factor IX is 0.1 ng or less Blood coagulation factor IX preparation derived from plasma . 血液凝固IX因子製剤の製造工程において、血液凝固IX因子に対する抗体を用いたイムノアフィニティークロマトグラフィー処理および疎水クロマトグラフィー処理を行うことによって得られる血漿由来の血液凝固IX因子製剤であって、該疎水クロマトグラフィー処理は、1Mよりも高く1.5M以下のNaCl濃度およびpH6以上でpH7よりも低いpHで血液凝固IX因子を疎水クロマトグラフィー担体に吸着させ、1Mよりも高く1.1M以下のNaCl濃度およびpH6以上でpH7よりも低いpHで担体を洗浄するものであり、血液凝固IX因子1国際単位当たりのMASP-1の含有量を0.1ng以下まで低減することで、最終産物での血液凝固IX因子に対する活性化血液凝固IX因子の含有率を0.1%以下としたことを特徴とする、血液凝固IX因子製剤。A plasma-derived blood coagulation factor IX preparation obtained by performing immunoaffinity chromatography treatment and hydrophobic chromatography treatment using an antibody against blood coagulation factor IX in the production process of the blood coagulation factor IX preparation, The chromatographic treatment is performed by adsorbing blood coagulation factor IX to a hydrophobic chromatography carrier at a NaCl concentration of higher than 1M and lower than 1.5M and a pH higher than 6 and lower than pH 7, and a NaCl concentration higher than 1M and lower than 1.1M. The carrier is washed at a pH of 6 or more and lower than pH 7. By reducing the content of MASP-1 per international unit of blood coagulation factor IX to 0.1 ng or less, blood coagulation IX in the final product is obtained. A blood coagulation factor IX preparation characterized in that the content of activated blood coagulation factor IX relative to the factor is 0.1% or less.
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