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

Description

  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 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.

  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.

  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).

Michaelski C.I. Vox Sanguinis, 55, No. 4, 1988, p. 202-210 Kamiya Tadashi, Journal of Japanese Society of Blood Transfusion, Vol. 33, No. 5, 1987, p. 638-644 Junichi Yasuda, Blood Products, Modern Publishing, 1986, p. 184-191 Burnow T. Et al., Journal of Biochemical and Biophysical Methods, 49, 1-3, 2001, p. 575-586 Josic D. Et al., Journal of Chromatography, 632, 1-2, 1993, p. 1-10 Burnow T. Et al., Vox Sanguinis, 57, 4, 1989, p. 225-232 Wataru Morikawa et al., Fundamental and Clinical, 24, 10, 1990, 713-717 Iga Y. Et al., Proceedings of the Sixth International Symposium on Hemophilia Treatment, Tokyo, Japan, 1990, p. 107-116 Akimoto Y. et al. Et al., Proceedings of the Sixth International Symposium on Hemophilia Treatment, Tokyo, Japan, 1990, p. 117-130

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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. 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.

  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. 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.

Claims (3)

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. 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 . 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.