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

Info

Publication number
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
Authority
JP
Japan
Prior art keywords
blood coagulation
coagulation factor
fix
masp
less
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
JP2005514403A
Other languages
Japanese (ja)
Other versions
JPWO2005033140A1 (en
Inventor
純一 下田
寿 矢野
美奈子 宮川
まい 東
洋一 緒方
高義 濱本
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chemo Sero Therapeutic Research Institute Kaketsuken
Original Assignee
Chemo Sero Therapeutic Research Institute Kaketsuken
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Chemo Sero Therapeutic Research Institute Kaketsuken filed Critical Chemo Sero Therapeutic Research Institute Kaketsuken
Priority to JP2005514403A priority Critical patent/JP4855074B2/en
Publication of JPWO2005033140A1 publication Critical patent/JPWO2005033140A1/en
Application granted granted Critical
Publication of JP4855074B2 publication Critical patent/JP4855074B2/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

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.
JP2005514403A 2003-09-30 2004-09-24 High-purity blood coagulation factor IX preparation and purification method thereof Expired - Fee Related JP4855074B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005514403A JP4855074B2 (en) 2003-09-30 2004-09-24 High-purity blood coagulation factor IX preparation and purification method thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2003339200 2003-09-30
JP2003339200 2003-09-30
JP2005514403A JP4855074B2 (en) 2003-09-30 2004-09-24 High-purity blood coagulation factor IX preparation and purification method thereof
PCT/JP2004/013905 WO2005033140A1 (en) 2003-09-30 2004-09-24 Highly pure blood coagulation factor ix preparation and method of purifying the same

Publications (2)

Publication Number Publication Date
JPWO2005033140A1 JPWO2005033140A1 (en) 2008-01-17
JP4855074B2 true JP4855074B2 (en) 2012-01-18

Family

ID=34419149

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2005514403A Expired - Fee Related JP4855074B2 (en) 2003-09-30 2004-09-24 High-purity blood coagulation factor IX preparation and purification method thereof

Country Status (2)

Country Link
JP (1) JP4855074B2 (en)
WO (1) WO2005033140A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110183906A1 (en) * 2008-04-24 2011-07-28 Celtic Pharma Peg Ltd. Factor ix conjugates with extended half-lives

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61263926A (en) * 1985-03-15 1986-11-21 ブリティッシュ・テクノロジー・グループ・リミテッド Factor ix protein
JPH04295432A (en) * 1991-01-25 1992-10-20 Immuno Ag Complex containing blood coagulation factor ix
WO1996027003A1 (en) * 1995-02-25 1996-09-06 Octapharma Ag A process for the preparation of factor ix from biological sources

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3877529T2 (en) * 1987-10-23 1996-11-07 Centre Regional De Transfusion Production of highly purified human factor IX and other plasma protein concentrate and its therapeutic use.
AT405608B (en) * 1997-04-08 1999-10-25 Immuno Ag METHOD FOR INACTIVATING PATHOGENS, ESPECIALLY VIRUSES, IN A BIOLOGICAL MATERIAL

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61263926A (en) * 1985-03-15 1986-11-21 ブリティッシュ・テクノロジー・グループ・リミテッド Factor ix protein
JPH04295432A (en) * 1991-01-25 1992-10-20 Immuno Ag Complex containing blood coagulation factor ix
JPH08259449A (en) * 1991-01-25 1996-10-08 Immuno Ag Preparation of pharmaceutical preparation containing hemo-coagulation ix factor
WO1996027003A1 (en) * 1995-02-25 1996-09-06 Octapharma Ag A process for the preparation of factor ix from biological sources

Also Published As

Publication number Publication date
WO2005033140A1 (en) 2005-04-14
JPWO2005033140A1 (en) 2008-01-17

Similar Documents

Publication Publication Date Title
JP5577005B2 (en) Thrombin preparation and production method thereof
AU2005228945B2 (en) Factor IXa for the treatment of bleeding disorders
EP2504349B1 (en) Method of purifying pegylated proteins
JP2012211189A (en) Use of medicine of factor-h concentrate
JPH04211372A (en) Improved method for production of thrombin and highly pure thrombin preparation obtained thereby
JP4445386B2 (en) Method for producing a virus-inactivated thrombin preparation
JP5261478B2 (en) Method for preparing factor X, activated factor X, inactive factor X and inactivated factor Xa, and pharmaceutical composition containing said factor
JPH0424360B2 (en)
JP5196799B2 (en) Ultra high purity FVIIa therapeutic preparation and method for obtaining the therapeutic preparation
RU2142806C1 (en) Method of purification and storage of, aqueous solution of purified factor ix, composition containing factor ix, and method of treatment
JPH1059866A (en) Production of blood coagulation factor vii and/or activated blood coagulation factor vii
SK4022000A3 (en) PHARMACEUTICAL SUBSTANCE CONTAINING VARIOUS VITAMIN K-DEPENDENTì (54) FACTORS
JPH053480B2 (en)
JP3043558B2 (en) Preparation of human activated protein C and method for its preparation
JP4855074B2 (en) High-purity blood coagulation factor IX preparation and purification method thereof
JP2014501721A (en) Methods for reducing and / or removing FXI and FXIa from solutions containing these coagulation factors
KR100377279B1 (en) How to make thrombin
JPH1052267A (en) Activation of prothrombin and production of thrombin based on activation thereof
JPH0733799A (en) Thrombin having high specific activity and its production
JP4105249B2 (en) Method for producing α2 plasmin inhibitor
JPH0329390B2 (en)
JPH04365481A (en) Production of thrombin
JPH02247199A (en) Production of human blood coagulation factor xiii
ES2792473T3 (en) Method to purify PEGylated proteins
Vogel et al. Preparation of modified bovine factor VIII with enhanced biological activity using insoluble-trypsin columns

Legal Events

Date Code Title Description
A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20100202

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20100405

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20100427

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20100628

A02 Decision of refusal

Free format text: JAPANESE INTERMEDIATE CODE: A02

Effective date: 20110315

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20110614

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A821

Effective date: 20110720

A911 Transfer of reconsideration by examiner before appeal (zenchi)

Free format text: JAPANESE INTERMEDIATE CODE: A911

Effective date: 20110811

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20111018

A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20111026

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20141104

Year of fee payment: 3

R150 Certificate of patent or registration of utility model

Free format text: JAPANESE INTERMEDIATE CODE: R150

Ref document number: 4855074

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R150

S531 Written request for registration of change of domicile

Free format text: JAPANESE INTERMEDIATE CODE: R313532

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

S531 Written request for registration of change of domicile

Free format text: JAPANESE INTERMEDIATE CODE: R313531

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

S111 Request for change of ownership or part of ownership

Free format text: JAPANESE INTERMEDIATE CODE: R313113

R350 Written notification of registration of transfer

Free format text: JAPANESE INTERMEDIATE CODE: R350

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

LAPS Cancellation because of no payment of annual fees