JP2003096094A - Purification method of protein S and preparation of protein S with high specific activity - Google Patents

Abstract

(57) [Summary] [Problem] Purification of protein S from plasma is extremely difficult due to the presence of many types of contaminants having a similar structure. A column chromatography method using an anti-protein S antibody was developed, and protein S with a very high purity was obtained by a simple operation.
However, it was not possible to exclude contamination by non-specific binding. The above problem was solved by subjecting a protein S-containing crude fraction from plasma to column chromatography in which an anti-protein antibody was immobilized on a carrier, and then to anion exchanger column chromatography in a subsequent step. .

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for purifying protein S and a protein S preparation having high specific activity due to activated protein C cofactor activity.

[0002]

2. Description of the Related Art Human protein S is contained in human plasma at about 10%.
It is a vitamin K-dependent plasma glycoprotein contained in mg / liter, and its molecular weight is about 80,000. The N-terminal region of human protein S has a structure very similar to that of vitamin K-dependent glycoproteins such as prothrombin, blood coagulation factors VII, IX and X, protein C and protein Z, but the C-terminal region Has a completely different structure from them, and in that region there is a sex hormone binding region,
The C4bp binding region and the blood coagulation factor V binding region are included. From this structural characteristic, it is considered that human protein S has many actions other than the blood coagulation system, unlike other vitamin K-dependent blood coagulation factors.

[0003]

[Problems to be Solved by the Invention] As a conventional method for purifying protein S, a method combining a barium precipitation method, an ammonium sulfate fractionation method, an anion exchanger treatment and the like (Prep. Biochem. 13, 1
91, (1983), Biochem.J. 209, 837 (1983), Throm
b.Res. 59, 759, (1990), etc.), but not only the concentration of human protein S in blood is very low, but also prothrombin, blood coagulation factor VII, factor IX, factor X, protein Since separation from C and protein Z and related proteins is not easy, it is difficult to collect and purify human protein S from blood in good yield by these methods. In recent years, RG Di by conventional chromatography
Method of Scipio et al. (Biochem. 16, 698-706 (1977)),
R. Jenny et al. (Prep. Biochem. 16, 227-245 (198
6)) and Koike's method (Japanese Patent Publication No. 7-59600) are reported. However, the method of RG Di Scipio et al. Is extremely complicated and unsuitable for mass production, and although the method of R. Jenny et al. And Koike et al. Using a monoclonal antibody are simple methods, human protein S is used as an antibody. Chaotropic ions must be used when eluting from the column,
There is a risk of denaturation of the desired human protein S.

Further, in JP-A-9-286799, there is proposed a method by chromatography using an adsorbent in which a monoclonal antibody specifically recognizing only the three-dimensional structure of protein S carrying a divalent metal ion is immobilized on an insoluble carrier. Has been done. This method elutes proteins under mild conditions by simply using a metal chelating agent, without using a liquid that may denature proteins such as chaotropic ions as an eluent, to obtain highly pure protein S. It can be recovered in high yield. This method is excellent in that the degree of purification is greatly increased in separating Protein S from related proteins, but in reality, it is due to non-specific binding to the column support itself. When contamination with impurities is unavoidable and a higher degree of purification is required, it is difficult to complete the purification only with the antibody column. On the other hand, the use of calcium ions sometimes activates blood coagulation factor II (prothrombin) to produce thrombin, and it may be inconvenient to use it as a drug as it is. Furthermore, protein S
Is susceptible to limited degradation by thrombin and the like in these purification processes, and protein S subjected to limited degradation loses the protein C cofactor activity. The production of protein S that has undergone this limited decomposition is a factor that reduces the protein C cofactor activity of protein S. For these reasons, a protein S preparation having a high specific activity of 50 international units or more per mg of total protein has not been obtained so far, using the protein C cofactor activity as an index.

[0005]

[Means for Solving the Problems] The present inventors carried out a relatively simple procedure by subjecting a crude fraction containing Protein S to anti-protein S antibody column chromatography and then to anion exchanger column chromatography. We succeeded in obtaining protein S with a high degree of purification even though it was a method. That is, according to the present invention, (1) in the step of purifying protein S from the crude fraction containing protein S, the crude fraction containing protein S is subjected to anti-protein S antibody column chromatography, and in the subsequent step, anion exchanger column chromatography is performed. Purification method of protein S to be subjected to chromatography, (2) The purification method according to (1), wherein the anti-protein S antibody is an antibody against human protein S, (3) The anti-protein S antibody is a monoclonal antibody against human protein S The purification method according to (1), (4) the anion exchanger column chromatography is a strong anion exchanger column chromatography, the purification method according to (1), (5) (1) to
(4) a purified protein S obtained by the purification method according to any one of (4), (6) a protein S preparation having a specific activity based on the activated protein C cofactor activity of 50 international units or more per mg of total protein, and ( 7) A pharmaceutical composition comprising the high specific activity protein S preparation according to (6) as an active ingredient.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION As the protein S-containing crude fraction used as a raw material for the purification method of the present invention, for example, the SI fraction and PIII fraction in the Kohn's ethanol fractionation method of human plasma are preferably used. The anti-Protein S antibody used for the antibody column is not particularly limited, but among them, an antibody that distinguishes and recognizes a three-dimensional structure due to the presence or absence of a divalent metal ion uses a chelating agent as an eluent. It is particularly preferable because it can be used under conditions. The monoclonal antibody recognizing only in the presence of the divalent metal ion and its preparation method are described in detail in JP-A-9-286799, and the outline thereof is as follows.

Hybridomas that produce monoclonal antibodies are obtained by the method of Koehler & Milstein.
Milstein; Nature: 256, 495-497 (1975)). That is, after immunizing a mouse with human protein S, the spleen cells of this mouse are fused with mouse myeloma cells. The obtained hybridoma is seeded in a microtiter plate, antibody screening and antibody-producing cell cloning are performed, and a monoclonal antibody is collected from this hybridoma cell. In the antibody screening, an antibody that recognizes both free and C4bp-bound human protein S only in the presence of a divalent metal ion is selected. When the obtained three-dimensional structure-specific monoclonal antibody in the presence of a divalent metal ion is used as an immunoadsorbent, the free conformation and C4b of the three-dimensional structure taken in the presence of a divalent metal ion are obtained.
It is possible to adsorb p-binding human protein S. By removing the adsorbed divalent metal ion bound to each human protein S with a metal chelating agent such as EDTA, each human protein S has a different three-dimensional structure from that when bound to the divalent metal ion. As a result, the affinity for the adsorbent disappears, and it is extremely easily eluted from the adsorbent. Therefore, highly purified free and C4bp-bound human protein S purified products without denaturation can be easily collected simultaneously in high yield.

The insoluble carrier used when an adsorbent is prepared by immobilizing or binding a monoclonal antibody to an insoluble carrier is generally used as a base material for immunoadsorption chromatography using a monoclonal antibody, a measuring reagent or a measuring kit. Any of those used for can be used. For example, cellulose, dextran, agarose, polyacrylamide, maleic acid polymer or a mixture thereof is preferably used as the material. The form of these inert carriers may be any form such as powder, granules, pellets, beads and fibers. In general, for a measurement kit, it is advantageous to use a plate having a large number of concave depressions (wells) used for measuring and separating plasma or its fractional components.

The above-mentioned adsorbent is used, and the free form and C
When the 4 bp binding type human protein S-containing mixture is contacted in the presence of a divalent metal ion, the monoclonal antibody immobilized on the adsorbent and, for example, calcium, magnesium, strontium, barium, manganese, etc.
It binds to human protein S, which has a three-dimensional structure only in the presence of a valent metal ion. In this way, if the adsorbent having human protein S having a three-dimensional structure specificity adsorbed thereto is treated with a chelating agent such as EDTA, EGTA, citrate, oxalate or phosphate, it will bind. Free divalent metal ions are removed and free and C
Each of the 4 bp-bound human protein S has a three-dimensional structure that can be formed only in the absence of a divalent metal ion, and the affinity for the adsorbent disappears. Therefore, an extremely mild eluent containing, for example, a chelating agent is used without using an eluent that may alter the antigen such as chaotropic ions, various protein denaturants or strongly acidic aqueous solution as in the conventional method. Thus, free and C4bp-bound human protein S without denaturation can be separated and purified very easily and in high yield.

The condition for loading the protein S-containing crude fraction on the antibody column is pH 6 to 8 by reacting with a buffer solution having a chelating effect such as citric acid or a divalent metal ion such as calcium ion such as phosphate. It is possible to use a substance having a buffering action in the vicinity of neutrality, such as Tris-hydrochloric acid buffer, except for a substance which produces an insoluble matter. Examples of the metal chelating agent that elutes the free and C4bp-bound human protein S having a three-dimensional structure in the presence of the divalent metal ion adsorbed on the antibody column from the column include EDTA, EGTA, citrate, and oxalate. Examples thereof include acid salts and phosphates.

The protein S after elution from the antibody column is contacted with an anion exchanger. Examples of this anion exchanger include those having an amino group as a functional group. Specifically, DEAE-Sepharose, DE
DEAE anion exchangers classified into weak anion exchangers such as AE-cellulofine and DEAE-Toyopearl, Q
Examples thereof include AE-Sephadex, Q-Sepharose, QAE Toyopearl, QA-Cellulofine, and POROS HQ, which are classified as strong anion exchangers. Of these, strong anion exchangers generally have better separation of impurities from protein S. The conditions for carrying out the chromatography using this anion exchanger are pH 6 to 8, and any buffer can be used as long as it shows a buffering property in the neutral region. To elute the free and C4bp-bound protein S adsorbed to the anion exchanger from the column, a buffer solution containing a salt such as sodium chloride at about 0.01 to 1.0 M is usually passed through. To be achieved. In particular, thrombin produced in the antibody column elutes at a salt concentration lower than that of protein S in the anion exchanger, so that separation is possible.
Proteins that bind nonspecifically to the antibody column behave differently in the anion exchanger depending on their types, but are often eluted at a salt concentration different from that of protein S, resulting in separation from protein S. It will be possible. Therefore, it was possible to further improve the degree of purification of protein S by treating with an anion exchanger after treatment with the antibody column.

A preferred procedure of the protein S purification method of the present invention will be described below. First, a crude fraction containing protein S, which is a raw material of the present invention, from human plasma, for example, SI of corn,
PIII and the like are prepared by a conventional method, and DEAE is used if necessary.
Pretreatment by treatment with an ion exchanger. A salt of a divalent metal is added to the obtained protein S solution to give a protein S having a three-dimensional structure only in the presence of a divalent metal ion. On the other hand, a column is filled with an adsorbent in which an antibody that recognizes only protein S having the three-dimensional structure is immobilized on a carrier, and this column is loaded with a solution containing a divalent metal ion and protein S. After washing if necessary, elution is performed with an eluent containing a chelating agent. Next, the eluate is loaded on the anion exchanger, washed if necessary, and then the protein S is eluted with the eluate. The obtained solution is concentrated to an appropriate concentration and subjected to liquid heat treatment for virus inactivation, freeze-drying and dry heating, and further virus removal membrane treatment if necessary. Human protein S obtained by such a series of operations contains almost no impurities, and the specific activity using the activated protein C cofactor activity shows a high specific activity of 50 international units or more per mg of total protein. Show. The obtained high specific activity human protein S preparation may be used as an active ingredient, and if necessary, other proteins such as albumin as a stabilizer and a substance having a physiological activity may be mixed to prepare a pharmaceutical composition.

[0013]

EXAMPLES The present invention will be described in more detail with reference to the following examples and test examples. The free type and C4bp binding type human protein S may be collectively referred to as human protein S. Example 1

(1) Preparation of an antibody column on which a three-dimensional structure-specific anti-human monoclonal antibody is immobilized Human protein S used as an antigen is the same as that described in R. G.
Purified according to the method of Di Scipio et al. Purified human
50 μg of protein S was dissolved in 50 μl of physiological saline, and 100 μl of complete Freund's adjuvant from Difco was added as an adjuvant to give a water-in-oil mineral oil emulsion as a basal immunizing antigen. As a booster immunization, 50 μg of the purified antigen was dissolved in 50 μl of physiological saline and used. Spleen cells obtained from a 7-week-old female Balb / c mouse immunized with a boosting antigen 60 days after the inoculation of the basal immunizing antigen were treated with mouse myeloma cells (SP2 / 0- After fusion with Ag14), cloning was performed to isolate a human protein S stereostructure-specific monoclonal antibody-producing hybridoma. A large number of hybridomas were prepared by proliferating the hybridoma cells in the abdominal cavity of mice to obtain a monoclonal antibody.

In order to measure the antigen specificity of the human protein S structure-specific monoclonal antibody thus obtained, human protein was analyzed by the same method as described in Example 1 of JP-A-9-286799. An enzyme-labeled antibody assay (ELISA) using S or various coagulation factors was performed to obtain a stereostructure-specific monoclonal antibody. On the other hand, 40 g of formyl cellulofine (manufactured by Seikagaku Corporation) was washed with 800 mL of distilled water on a glass filter, and further a coupling buffer (0.2 M phosphate buffer, pH 7.
0) Washed with 200 mL. Immediately after removing the coupling buffer by suction, the gel was immediately added to the coupling buffer solution of the monoclonal antibody obtained above (2.5 mg
/ ML) was suspended in 80 mL and gently shaken at 4 ° C. for 2 hours. After that, 10 mg / g Gel of sodium cyanoborohydride was added as a reducing agent, and further 4 ° C.
Then, it was shaken gently overnight. After removing the supernatant, the glass filter was washed with about 800 mL of distilled water and then 80 m of a coupling buffer solution containing 1 M ethanolamine.
Transfer to L and add sodium cyanoborohydride to 1
0 mg / g Gel was added, and the remaining active groups were blocked by shaking at 4 ° C. for 2 hours. After blocking, the gel was washed on a glass filter with distilled water and then thoroughly washed with 20 mM acetate buffer (pH 7.3), and packed in a column.

(2) Purification of protein S from human plasma 5 L of human plasma was loaded onto a DEAE-Sepharose column equilibrated with 20 mM citrate buffer (pH 7.0) in advance. 260m after washing with equilibration buffer
Fractions containing human protein S were obtained using equilibration buffer containing M sodium chloride. Final concentration of 2 in that fraction
Calcium chloride was added to 5 mM, and benzamidine hydrochloride was added to a final concentration of 1 to 10 mM,
The column was loaded on the anti-human protein S antibody column previously equilibrated with 20 mM acetate buffer (pH 7.3) containing 2 mM calcium chloride. After washing sequentially with an equilibration buffer containing 1 M sodium chloride and 150 mM sodium chloride, the protein S fraction was gently eluted with 20 mM acetate buffer (pH 7.3) containing 5 mM EDTA and 150 mM sodium chloride. Next, this fraction was preliminarily supplemented with 20 mM of 150 mM sodium chloride.
POROS equilibrated with mM acetate buffer (pH 7.3)
Loaded on a 50HQ column. After washing with an equilibration buffer containing 350 mM sodium chloride, elution was performed with an equilibration buffer containing 500 mM sodium chloride to obtain a fraction containing human protein S. The activated protein C cofactor activity of the obtained protein S fraction was measured (Roche
The yield in this step was 80% when the yield in this step was determined using Staclott Protein S manufactured by Diagnostics.

Example 2 An SI fraction was obtained from 5 L of human plasma by Cohn fractionation, and this was loaded on a DEAE-Sepharose CL-6B column equilibrated with 20 mM citrate buffer (pH 7.0) in advance. After washing with the equilibration buffer, elution was performed with an equilibration buffer containing 260 mM sodium chloride to obtain a human protein S-containing fraction. Next, using this fraction, antibody column treatment was carried out in the same manner as in Example 1. Then, the elution fraction of the antibody column was loaded onto the POROS 50HQ column as in Example 1 and eluted to obtain a human protein S fraction. Furthermore, this fraction is subjected to ultrafiltration (PALL FILT).
Desalination and concentration were carried out using OMEGA 10K membrane manufactured by RON. The protein content is from the absorbance at 280 nm,
It was calculated using a molecular extinction coefficient of 9.5 in a 1% protein S solution. The properties of the obtained Protein S preparation were as follows. Properties: colorless and clear protein content: 3.5 mg / mL protein S activity: 190.6 IU / mL specific activity: 54.5 IU / mg Test Example 1 Protein amount in a sample of a series of steps carried out in Example 2, The protein S activity and thrombin activity were measured.
As the protein amount, the absorbance at 280 nm by a spectrophotometer was used. The protein S activity was measured as an activated protein C cofactor activity using Staclott protein S manufactured by Roche Diagnostics. Thrombin activity is a fluorescent peptidic synthetic substrate 3093v
(Peptide Institute) was used to measure the hydrolytic activity of the synthetic substrate by thrombin using a fluorescence spectrophotometer. The results are shown in Table 1. After POROS 50HQ, an increase in protein S specific activity and a decrease in thrombin activity were confirmed.

[0018]

[Table 1]

Test Example 2 SDS of the solution after the antibody column of Example 2 (solution before POROS 50HQ) and the fraction after POROS 50HQ
The polyacrylamide gel electropherogram is shown in FIG. S
DS polyacrylamide gel electrophoresis was performed at an acrylamide concentration of 10%, and visualization was performed by a silver staining method. It was confirmed that impurities other than protein S found in the solution after the antibody column, which are considered to be nonspecific binding, were removed by the POROS 50HQ treatment.

[0020]

EFFECT OF THE INVENTION According to the purification method of the present invention, substances that have been contaminated by non-specific bindings other than protein S, which are present even after the antibody column treatment, are removed at once by the anion exchanger treatment, and the conventional method is used. Protein S, which has a high protein C cofactor specific activity, which could not be obtained.

[0021]

[Brief description of drawings]

FIG. 1 Comparison of purification degree before and after the POROS 50HQSDS polyacrylamide gel electrophoresis operation

[Explanation of symbols]

A: Before antibody column treatment B: After antibody column treatment

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hitoshi Tanaka             1 Nihon Pharmaceutical Co., Ltd., 3 Shinizumi, Narita City, Chiba Prefecture             Ceremony company Narita factory F-term (reference) 4C084 AA02 AA06 BA44 CA18 DC10                       NA20 ZA532                 4H045 AA10 AA20 AA30 CA40 DA76                       EA20 GA21 GA26

Claims (7)

[Claims] 1. A protein S-containing crude fraction is converted to protein S
In the step of purifying protein S, the protein S-containing crude fraction is subjected to anti-protein S antibody column chromatography, and in the subsequent step anion exchanger column chromatography is used to purify protein S. 2. An anti-protein S antibody is human protein S
The method according to claim 1, which is an antibody against 3. An anti-protein S antibody is human protein S
The method according to claim 1, which is a monoclonal antibody against 4. The purification method according to claim 1, wherein the anion exchanger column chromatography is a strong anion exchanger column chromatography. 5. A purified protein S obtained by the purification method according to any one of claims 1 to 4. 6. A high specific activity protein S preparation having a specific activity based on activated protein C cofactor activity of 50 international units or more per mg of total protein. 7. A pharmaceutical composition containing the high specific activity protein S preparation according to claim 6 as an active ingredient.