JP4046377B2 - Method for activating blood coagulation factor VII and method for producing activated blood coagulation factor VII based on the method - Google Patents

Description

【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１０倍カラム容量洗浄し、５０ｍＭＴｒｉｓ／３０ｍＭＮａＣｌ／２.０ｍＭＣａＣｌ₂緩衝液(ｐＨ８.０)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。
【００１６】
【表２】

【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、５０ｍＭＴｒｉｓ／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。
【００１８】
【表３】

【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-ｌｅｓｓＦＶＩＩaの産生が始まることを示している。
【００２０】
【表４】

【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(ｐＨ８.５)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。
【００２２】
【表５】

【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(ｐＨ８.５)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。
【００２４】
【表６】

【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、Ｃａ²⁺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。
【００２６】
【表７】

[0001]
[Industrial application fields]
The present invention relates to the field of plasma proteins. More specifically, the present invention relates to a method for producing activated blood coagulation factor VII (hereinafter sometimes referred to as FVIIa) on a plasma fraction containing FVII or an FVII-producing cell culture produced based on a gene recombination technique. The present invention provides a method for producing FVIIa, which can produce FVIIa with a reduced content of contaminating proteins very simply by treatment with anion exchange chromatography and activation in the liquid phase.
[0002]
[Background Art and Problems to be Solved by the Invention]
FVII is a vitamin K-dependent blood coagulation factor and is widely known to be an initiation factor for exogenous blood coagulation. Like other vitamin K-dependent coagulation factors, the amino acid sequence from the N-terminal to 35 residues has a Gla region consisting of 10 gamma carboxyglutamic acids (hereinafter sometimes referred to as Gla) (Proc. Natl. Acad. Sci. USA, vol. 83, p. 2412-2416, 1986). FVII is in vitro 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 FIXa). It is known that 152Arg-153Ile is hydrolyzed and converted into an active FVII or FVIIa composed of an H chain and an L chain cross-linked by a single S—S bond (J Biol. Chem., Vol. 251, p. 4797-4802, 1976). Recently, Saulius et al. Reported that the physiological FVII activating enzyme is activated blood coagulation factor X (Biochemistry, vol. 35, p. 1904-1910, 1996).
[0003]
As replacement therapy for hemophilia A and hemophilia B patients, administration of a blood coagulation factor VIII (hereinafter sometimes referred to as FVIII) and blood coagulation factor IX (hereinafter sometimes referred to as FIX) preparation is performed. It is done. However, the emergence of neutralizing antibodies against FVIII and FIX (sometimes referred to as inhibitors) has been regarded as a problem with the treatment methods. It has already been reported that FVIIa is effective in the treatment of such inhibitor patients. Currently, development of plasma-derived FVIIa and genetically modified FVIIa using genetic recombination technology is underway (basic and clinical). , vol. 30, p. 315-325, 1996; Haemostasis, vol. 19, p. 335-343, 1989)
[0004]
Various tests were performed on the conversion of the enzyme precursor FVII to FVIIa, and in the absence of FVII activating enzymes FXa, FIXa and FVIIa, FVII self-activation reaction occurs due to the involvement of FVIIa, That is, it has been clarified that the reaction proceeds by the reaction “FVII + FVIIa → 2FVIIa” represented by the following formula (Biochemistry, vol. 28, p.9331-9336, 1989).
By the way, as a method for preparing FVIIa, there is an activation method using an anion exchange resin (Res. Discl., Vol. 269, p. 564-565, 1986). Although details of the reaction are not clarified, this method is extremely effective for producing FVIIa on an industrial scale. However, a problem in the activation of FVII using an anion exchange resin is that a Gla region-deleted FVIIa (hereinafter referred to as GD-lessFVIIa) in which 38 amino acids have been deleted from the N-terminus of the L chain of FVII as a byproduct. May be called). FVIIa activity in vivo requires binding to phospholipids and tissue factors, and the Gla region is essential for this purpose. Therefore, GD-lessFVIIa, which does not have a Gla region, has very weak binding to phospholipids and tissue factors, and 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. 679-685, 1992).
[0005]
Up to now, activation on anion exchange resin has been achieved by developing FVII on the gel, then completely activating it, and elution with a gradient, thereby utilizing the difference in isoelectric point between GD-lessFVIIa and FVIIa as a by-product at the elution stage. In the activation reaction by anion exchange chromatography using MonoQ, Thim et al. Stated that produced GD-lessFVIIa can be separated (Biochemistry, vol. 27, p. .7785-7793, 1988). However, in practice, the complete separation is extremely difficult, and the yield between processes is significantly reduced if the separation is performed strictly. Furthermore, there has been no report on the production mechanism of GD-lessFVIIa on an anion exchange resin so far, and it has been extremely difficult to control the production on an anion exchange resin.
[0006]
[Means for Solving the Problems]
In view of the above problems in the activation reaction, the present inventors have conducted extensive studies to suppress the production of GD-lessFVIIa, which is a by-product in the activation reaction on the anion exchange resin, The present invention has been completed.
In the present invention, the activation reaction on the anion exchange resin is suppressed to less than 60%, and the eluted FVII / FVIIa mixed solution is aged 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. When the present invention is applied, production of GD-lessFVIIa generated in the conversion of FVII to FVIIa in the activation process is suppressed, and a remarkable increase in inter-process yield is possible.
[0007]
Hereinafter, the details of the present invention will be described.
The starting material used in the present invention is: (1) FVII-containing solution prepared by using an appropriate chromatographic operation or corn ethanol separation method or an improved method thereof, or (2) gene recombination technology. FVII and / or FVII derivative-containing solutions prepared from FVII-producing transformed cells produced based on the above are targeted, but the optimal starting material is a protein having a Gla region obtained by roughly purifying plasma by anion chromatography The solution (PPSB fraction) is developed on an anti-FVII monoclonal antibody-immobilized affinity gel and contains almost only FVII eluted. The purification of FVII is not particularly limited to a method based on the principle of immunoadsorption, and any method that can purify FVII may be used.
[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, but 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, and the contact with the ligand can be carried out by a batch method or a continuous column method. As an optimal aspect, the above anion exchange resin is used in a chromatography column. And the adsorbed desired FVII is eluted. Elution from anion exchange resin is activated to FVIIa by using stepwise elution method (stepwise elution method) with a buffer containing Ca ²⁺ of 0.5 mM or more. Elute so that the rate is 60% or less.
In order 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, the increase in the FVII development amount in the resin and the residence time in the resin increases the activation rate, and the decrease in the FVII development amount in the resin and the residence time in the resin decreases the activation rate.
[0009]
The aging time after elution is determined by (1) the mixing ratio of FVII / FVIIa at the time of elution and (2) the protein concentration of FVII / FVIIa. 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. The amount is preferably 1.0 to 3.0 mg / ml, and the amount developed on the anion exchange resin and the column size are determined as such. Further, the activation rate at the time of elution may be controlled by the residence time in the resin as described above if the amount of FVII developed on the anion exchange resin is determined. 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, production of GD-lessFVIIa becomes remarkable, which is not appropriate. Preferably a concentration range of 1.0 to 10 mM is used. When the activation rate of FVIIa in the mixed solution of FVII / FVIIa exceeds 95%, the protein concentration is decreased by dilution operation to delay the activation reaction rate, or the pH is lowered to reduce the enzyme reaction. This is done by pausing. The obtained FVIIa solution may be formulated into a desired pharmaceutical preparation by dialysis.
[0010]
For formulations for intravenous administration, the composition is usually in an aqueous solution containing a physiologically compatible substance such as sodium chloride, glycine and the like and having a buffered pH that is compatible with physiological conditions. Dissolve in Further, from the viewpoint of ensuring long-term stability, it may be considered to take the form of a lyophilized preparation as the final dosage form. It should be noted that guidelines for intravenously administered compositions are established by government regulations, such as “Biological Formulation Standards”.
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[0011]
【Example】
Prior to the description of the examples, the method for 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. In the FVII quantification method, a specimen is added to FVII-deficient plasma, incubated for a certain period of time, a PT reagent containing tissue factor, phospholipid, and Ca ²⁺ is added, and the clotting time is calculated.
2) Measurement of FVIIa content SDS-PAGE is used for the measurement of FVIIa content. When activated, FVII (molecular weight 50 kda) divides 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. In the reduced SDS-PAGE, the inactive substance is confirmed at a molecular weight of 50 kda, the H chain at 30 kda, and the L chain at 20 kda. The detected band was read with a densitometer, the band having a molecular weight of 50 kda was defined as unactivated FVII content%, and the sum of the H chain and L chain contents 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]
Example 1
The supernatant obtained by cold thawing 100 liters of fresh frozen plasma and centrifuging the precipitate fraction was passed through an anion exchanger (DEAE-Sephadex A-50: Pharmacia) column, and 20 mM citric acid / 0.1 M NaCl buffer. The solution was sufficiently washed with a solution (pH 7.0), and the PPSB fraction having the Gla domain was eluted with 20 mM citric acid / 0.5 M NaCl buffer (pH 7.0).
Ten liters of the eluate was developed on an anti-FVII monoclonal antibody immobilized affinity gel pre-equilibrated with 50 mM Tris / 150 mM NaCl / 5.0 mM CaCl ₂ buffer (pH 8.0), and 50 mM Tris / 2.5 M NaCl / 5.0 mM CaCl ₂ buffer ( After washing with pH 8.0), it was further washed with 50 mM Tris / 30 mM NaCl / 5.0 mM CaCl ₂ buffer (pH 8.0) and eluted with 50 mM Tris / 30 mM NaCl / 10 mM EDTA buffer (pH 7.4) to obtain the FVII fraction. . The FVII fraction was developed on a virus removal membrane (Bemberg microporous membrane, Asahi Kasei) that was previously filled with 50 mM Tris / 30 mM NaCl buffer (pH 8.0) to obtain a filtrate. The FVII purity of the obtained filtrate was 85%.
[0013]
50 ml of the above 0.2 mg / ml FVII solution was lined on a DEAE-Sepharose Fast Flow (Pharmacia) column having an inner diameter of 5.0 mm and a height of 5.0 cm, which had been equilibrated in advance with 50 mM Tris / 30 mM NaCl buffer (pH 8.0). The plate was developed at a speed of 300 cm / hr, washed with various column volumes with the above buffer under the same linear speed, and eluted with 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 shown in Table 1 were obtained. This result shows that when the washing column volume exceeds 70 column volumes, the activation rate of FVIIa exceeds 66.4% and production of GD-lessFVIIa starts.
[0014]
[Table 1]

[0015]
Example 2
50 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 equilibrated in advance with 50 mM Tris / 30 mM NaCl buffer (pH 8.0), and the inner diameter was 5.0 mm and the height was 5. The column was developed on a 0 cm DEAE-Sepharose Fast Flow (Pharmacia) column at a linear velocity of 300 cm / hr, and further washed 10 times with the above buffer solution at the same linear velocity, and 50 mM Tris / 30 mM NaCl / 2.0 mM CaCl ₂ buffer ( The solution was eluted at various linear speeds using pH 8.0). SDS-PAGE analysis of the eluted FVII / FVIIa fraction was performed and the results shown in Table 2 were obtained. This result indicates that when the elution linear velocity is less than 100 cm / hr, the activation rate of FVIIa exceeds 62.8%, and production of GD-lessFVIIa starts.
[0016]
[Table 2]

[0017]
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 equilibrated in advance with 50 mM Tris / 30 mM NaCl buffer (pH 8.0), and the inner diameter was 5.0 mm and the height was 3. The column was developed on a 3 cm DEAE-Sepharose Fast Flow (Pharmacia) column at a linear speed of 200 cm / hr, and further washed with 10 column volumes with the above buffer under the same linear speed, and then 50 mM Tris / 30 mM NaCl / 1.75 mM CaCl ₂ buffer (pH 8). 0.0) at a linear velocity of 150 cm / hr. The eluted FVII / FVIIa mixed solution having a concentration of 2.0 mg / ml was further aged in the liquid phase for 10 hours. The results immediately after column elution (before aging) and after aging are shown in Table 3.
As a result, GD-lessFVIIa was not produced when the activation rate after elution was 60% or less even under conditions where the development amount to an anion exchange resin and the column size were different from those of Examples 1 and 2, and even after aging. It shows that its production is extremely suppressed.
[0018]
[Table 3]

[0019]
Example 4
35 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 equilibrated in advance with 10 mM Tris / 100 mM NaCl buffer (pH 8.5), and the inner diameter was 5.0 mm and the height was 5. Developed on a 0-cm Q-Sepharose Fast Flow (Pharmacia) column at a linear speed of 300 cm / hr, and further washed with various column volumes with the above buffer at the same linear speed, and then washed with 50 mM acetate buffer to deactivate the adsorbed FVIIa. Elute with liquid (pH 3.5). SDS-PAGE analysis of the eluted FVII / FVIIa fraction was performed and the results shown in Table 4 were obtained. This result shows that the activation rate of FVIIa exceeds 61.3% and the production of GD-lessFVIIa starts when the washing column volume exceeds 40 column volumes.
[0020]
[Table 4]

[0021]
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 equilibrated in advance with 10 mM Tris / 100 mM NaCl buffer (pH 8.5), an inner diameter of 5.0 mm, and a height of 5. The column was developed on a 0-cm Q-Sepharose Fast Flow (Pharmacia) column at a linear velocity of 300 cm / hr, and further washed with 10 times the column volume with the above buffer solution at the same linear velocity, and 10 mM Tris / 100 mM NaCl / 4.0 mM CaCl ₂ buffer solution ( Elution was performed at various linear speeds using pH 8.5). SDS-PAGE analysis of the eluted FVII / FVIIa fraction was performed, and the results shown in Table 5 were obtained. This result shows that when the elution linear velocity is 108 cm / hr or less, the activation rate of FVIIa exceeds 62.7% and the production of GD-lessFVIIa starts.
[0022]
[Table 5]

[0023]
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 equilibrated in advance with 10 mM Tris / 100 mM NaCl buffer (pH 8.5), an inner diameter of 5.0 mm, and a height of 5. The column was developed on a 0 cm Q-Sepharose Fast Flow (Pharmacia) column at a linear velocity of 300 cm / hr, and further washed with 10 column volumes with the above buffer under the same linear velocity, and then 10 mM Tris / 100 mM NaCl / 4.0 mM CaCl ₂ buffer (pH 8). And elution 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. The results immediately after column elution (before aging) and after aging are shown in Table 6.
As a result, even when Q-Sepharose Fast Flow (Pharmacia) was used as an anion exchange resin as in Example 3, GD-lessFVIIa was not produced when the activation rate after elution was 60% or less. It shows that its production is extremely suppressed even after liquid aging.
[0024]
[Table 6]

[0025]
Example 7
In the same manner as in Example 3, after partial activation using DEAE-Sepharose Fast Flow (Pharmacia), 5.0 mg / ml containing 2.0 mM of Ca ²⁺ concentration aged in the liquid phase and fully activated. The FVIIa mixed solution at a concentration was adjusted to 50 mM Tris / 30 mM NaCl buffer so that the Ca ²⁺ concentration was 0.17 mM, 0.50 mM, 0.75 mM, 1.0 mM, 2.0 mM, and the protein concentration was 0.4 mg / ml. The mixture was diluted with pH 8.1) and incubated at 9 ° C. for 88 hours.
The results after incubation are listed in Table 7. The results in Table 7 indicate that the optimal Ca ²⁺ concentration for GD-lessFVIIa production is less than 0.5 mM, and the degradation reaction of FVIIa to GD-lessFVIIa is inhibited under a Ca ²⁺ concentration of 0.75 mM or more. It also shows that This result shows the threshold of Ca ²⁺ concentration of GD-lessFVIIa production reaction in the liquid phase, and when elution from an anion exchange resin is carried out with a buffer containing Ca ²⁺ , production of GD-lessFVIIa at the time of elution is shown. This indicates that the Ca ²⁺ concentration in the elution buffer is required to be 0.5 mM or more for inhibition.
[0026]
[Table 7]

Claims (3)

  (a) A solution containing blood coagulation factor VII (hereinafter sometimes referred to as FVII) is developed and brought into contact with an anion exchange resin, and (b) FVII is active while maintaining an activation rate of less than 60%. Activated to coagulated blood coagulation factor VII (hereinafter sometimes referred to as FVIIa), and (c) eluted with an elution buffer containing Ca2 + at a concentration of 0.5 mM or more, (d) the eluate A method of activating blood coagulation factor VII to activated blood coagulation factor VII through a step of leaving (aging) the solution in a solution state to completely activate the inactive molecules.   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). A method for producing activated blood coagulation factor VII, comprising a step based on the method for activating FVII according to claim 1.
more than