JP2001031698A - Purified antithrombin-iii and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide purified antithrombin-III not having fear of viral infection and not containing substantially cloven antithrombin-III having fear about the side effect. SOLUTION: This purified antithrombin-III substantially not containing active virus and cloven antithrombin-III is obtained by subjecting human plasma or a derived human plasma fraction to a purification process at a temperature not higher than 130 deg.C using immobilized heparin, by subjecting the human plasma or the derived human plasma fraction to a drying process and by heating the dried antithrombin-III until the virus is inactivated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a purified antithrombin-III substantially free from cleaved antithrombin-III, which has no risk of virus infection and is likely to cause side effects, and a method for producing the same.

[0002]

2. Description of the Related Art Antithrombin-III (AT-III) has a molecular weight of 65, belonging to α2 globulin present in plasma.
It is a kind of glycoprotein of 000 to 68,000 and has protease inhibitory activity and strongly inhibits thrombin clotting activity. Coagulation factors other than thrombin, such as activated X
It also has an inhibitory effect on factors, activated factor IX, and the like. Others have been reported to have an inhibitory effect on plasmin and trypsin. It is known that these inhibitory effects generally progress more rapidly in the presence of heparin. AT-III having such an effect is used for correcting hypercoagulability, specifically for treating various diseases caused by generalized intravascular coagulation syndrome (DIC) and a decrease in AT-III level in blood. Used. Since AT-III is a protein present in plasma, it is essential to incorporate a step of inactivating a virus that may be contaminated when using it as a therapeutic drug. Conventional A
The virus inactivation treatment by heating an AT-III-containing aqueous solution used in the production of T-III preparations also has a negative effect on proteins, thereby denaturing or inactivating AT-II.
I generate. Therefore, a purification method including a purification step using immobilized heparin after the virus inactivation step (JP-A-6
3-23896), a method of removing impurities by hydrophobic chromatography (Japanese Patent Laid-Open No. 1-275600), AT-
III Add the selected stabilizer during the heat treatment of the aqueous solution,
A method of maintaining the ratio of the AT-III monomer at a certain level or higher (Japanese Patent Application Laid-Open No. H10-147538) and a method of treating with a metal chelate resin (Japanese Patent Application Laid-Open No. H11-49799) have been proposed.

[0003]

However, even after the AT-III-containing aqueous solution is heat-treated for virus inactivation, the purification method using immobilized heparin or other purification methods described above is still carried out. Contains a non-negligible amount of denatured or inactive AT-III. Recent studies have revealed that most of this denatured or inactive AT-III has reduced affinity for heparin. The present inventors have found, among the denatured or inactive AT-IIIs having a low heparin affinity, those showing a lower mobility than the main peak AT-III on SDS polyacrylamide gel electrophoresis. As a result of taking out and performing N-terminal partial amino acid sequence analysis, at least Arg
AT-II cleaved at and around 393-Ser394
I found out that I was. The cleaved AT-III is likely to be a substance that has been conventionally concerned with the induction of neutrophil migration and the relationship with the release of cytokines in cell lines. Modified or inactive AT with low heparin affinity
The removal of -III is difficult by purification with immobilized heparin, and since they have very similar chemical structure and physical properties to the native AT-III, if they are strictly removed, the yield is low. I cannot help but drop.

[0004]

The present inventors have found that once denatured or inactive AT-III with low heparin affinity is produced, it can be separated and removed from native AT-III. In view of the fact that it is extremely difficult, denatured or inactive AT-III with low heparin affinity, especially cleaved AT
Various studies were conducted on methods for suppressing the production of -III and inactivating the virus. As a result, it was obtained by drying AT-III purified with immobilized heparin or the like at a low temperature of 13 ° C. or less at a temperature as low as possible, and heating the dried AT-III until the virus was inactivated. Purified AT-III is the active virus and the cleaved A
They found that they did not substantially contain T-III, and further studied to complete the present invention. That is, the present invention
(1) purified AT-III substantially free of active virus and cleaved AT-III; (2) human plasma or induced human plasma fraction subjected to a purification step using immobilized heparin at a temperature not exceeding 13 ° C. And further subjecting the dried AT-III obtained to a drying step to heating the dried AT-III until the virus is inactivated, and purified AT-III substantially free of cleaved AT-III. (3) After subjecting to a purification step using immobilized heparin, before the drying step, the AT
The method for producing purified AT-III according to the above (2), in which the solution containing -III is subjected to an adsorption / desorption step using a strong anion exchanger and a purification step using immobilized heparin, and (4) virus inactivation, The method for producing purified AT-III according to the above (2), which is performed at 80 ° C. for 24 to 120 hours.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The raw material used in the present invention is a human plasma or a human plasma fraction containing human AT-III, which can be obtained, for example, by a human plasma corn low-temperature ethanol fractionation method. Supernatant I, supernatant II +
III, fraction IV-1, blood coagulation VII from citrate-containing plasma
Fractions derived from human plasma, such as the residual fraction after recovery of factor I, may be mentioned. Of these, supernatant I is a particularly preferred raw material. In the present invention, the raw human plasma or the induced human plasma fraction is first used at 13 ° C. or lower, preferably at 0 ° C.
At 10 ° C., it is subjected to a purification method using immobilized heparin, ie, heparin affinity chromatography. This step itself is known, and the adsorption support suitably used in the present invention is, for example, one in which heparin is covalently bonded to an insoluble carrier such as cellulose or agarose. By this step, most of the contaminants can be removed.
The obtained solution containing AT-III is subjected, if necessary, to ultrafiltration known per se, and further to an adsorption / desorption treatment step using a strong anion exchanger, if necessary. These steps are also 13
C. or lower, preferably at 0 to 10C. The strong anion exchanger used has a dissociation degree (pKa) of usually 10.3 or more, preferably 10.5 or more. Those having a quaternary amino group, particularly those having a trialkylaminoalkyl group are preferably used as structural features. Specific examples of the strong anion exchanger include those having a trialkylaminoalkyl group such as a trimethylaminomethyl group and a diethylhydroxypropylaminoethyl group. Among these, those having a trimethylaminomethyl group are particularly preferred.

Examples of the insoluble carrier having these functional groups include cellulose, agarose, dextran,
Examples include polyacrylamide, amino acid copolymer, polyvinyl copolymer, and polystyrene copolymer. AT
-III-containing solution is contacted with a strong anion exchanger at pH 6
８8 and a salt concentration of about 0-0.5 M are preferable. As a solvent having such a condition, for example, 0.05 M
0.01M phosphate buffer containing sodium chloride (pH 6.5)
To 7.5). The elution conditions are pH 6 ~
It is preferable to have a salt concentration of about 8 and a salt concentration of about 0.1 to 0.5 M, and specific examples thereof include a 0.01 M phosphate buffer (pH 7) containing 0.17 M sodium chloride as a solvent.
The contact method may be either a column method or a batch method. When the batch method is used, the AT-III prepared under the above contact conditions is used.
The aqueous solution containing is contacted with the strong anion exchanger equilibrated under the same conditions. The condition is that the exchanger 1 ml
The mixture is mixed with 1 to 100 ml of the aqueous solution for about 30 minutes to 2 hours, and then centrifuged to collect the exchanger. Further, the above-mentioned elution solvent is added to the exchanger. The mixing conditions are the same as for the contact, but the supernatant is recovered by centrifugation. On the other hand, when the column method is used, the aqueous solution containing AT-III prepared under the above-mentioned contact conditions is equilibrated under the same conditions, and passed through the strong anion exchanger packed in the column, and the non-adsorbed fraction is discarded. I do. After washing the column if necessary, the elution solvent is allowed to flow to collect the eluted fraction.

[0007] By this step, heparin could not be completely removed even by affinity chromatography.
Contaminants having an affinity for heparin and various viruses that may be present are substantially removed. Thereafter, if necessary, the above purification step using immobilized heparin may be performed again. The purified AT-III thus obtained
The aqueous solution containing is dried so as to have a water content of 5% or less, preferably 3% or less. Also in this drying step, the treatment temperature is preferably as low as possible, and this condition is satisfied by ordinary freeze-drying. The dried composition may be in the form of a powder, a cake, or any other form. The obtained dry AT-III is heated until the virus is inactivated. The heating temperature is 50 to 80C, preferably 55 to 75C, more preferably 60 to 70C, and the heating time is 24 to 120 hours, preferably 72 to 120C.
It is about 108 hours, more preferably about 84 to 100 hours. As the atmosphere at the time of heating, air at atmospheric pressure is usually used, but if necessary, it may be carried out in reduced pressure air, nitrogen or other inert gas. For the purpose of minimizing thermal denaturation of AT-III during heating, sugars, sugar alcohols, various amino acids, organic acid salts, inorganic acid salts, etc. are added to the solution, and then dried and subjected to heat treatment. May be attached. Heating may be performed by any means, but a means such as an oven, infrared irradiation, a sand bath or a water bath is appropriately used.

[0008] By the operation of this heating step, the virus is inactivated and, unlike in the case of liquid heating, the cleaved AT
A purified AT-III substantially free of -III is obtained. Here, AT-II containing substantially no active virus
I means AT-III in which the virus has been reduced to a level at which the virus cannot be cultured by the inactivation treatment of the virus. AT-III that has reached this state is not at risk of virus infection even when administered to humans. AT-III substantially free of cleaved AT-III means that the cleaved AT-III was not recognized as a result of the N-terminal partial amino acid sequence analysis of Test Example 2 described later. In addition, by performing a virus removal membrane treatment or the like during such a purification step, a safer preparation can be provided. Examples of the preparation include an aqueous solution, a suspension, and a lyophilized preparation. These preparations are appropriately mixed with pharmaceutically acceptable additives (diluent, isotonic agent, surfactant, etc.) It can be carried out by conventional means on the formulation. The preparation is used as an injection such as intravenous injection, and the lyophilized preparation is used by dissolving it in a solution such as distilled water for injection at the time of use. The highly purified AT-III obtained according to the present invention can be used for generalized intravascular coagulation syndrome (DIC)
In addition, after surgery, it can be administered to patients whose blood AT-III level has decreased due to hepatitis, liver cirrhosis, pancreatitis, neonatal respiratory distress syndrome, etc., to prevent thrombus formation.

[0009]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Example 1 After treating about 17.5 liters of the supernatant I of the corn low-temperature ethanol fraction with a DEAE anion exchanger, the unadsorbed fraction was collected as a pool. 0.02M phosphate buffer (pH
Heparin-Sepharose 6F previously equilibrated in 7.3)
The above pool fraction was loaded at 5 ° C. onto a column packed with about 370 ml of F gel. Subsequently, after washing with 3.0 liters of a 0.02 M phosphate buffer and 0.3 M sodium chloride solution (pH 7.3) at 5 to 10 ° C., respectively, a 0.02 M phosphate buffer and a 2.0 M chloride, respectively. AT-III was eluted with 3.0 liter of sodium solution (pH 7.3).
Then, the AT eluted from the heparin-Sepharose gel
The -III fraction was desalted using an ultrafiltration device (pore size 10K, manufactured by Filtron) at 5 to 10 ° C to a salt concentration of 0.1 M or less, and concentrated to about 200 ml. The above fraction was subjected to 0.01M phosphate buffer (pH 7.0).
To about 1.0 liter. Q-Sepharose F previously equilibrated with 0.01 M phosphate buffer (pH 7.0)
F gel (Pharmacia, Trimethylaminomethyl /
Crosslinked agarose) 5 to 1 of the above-mentioned diluent was added to about 340 ml.
Loaded at 0 ° C. Then, under the same temperature conditions, a 0.01 M phosphate buffer solution and a 0.12 M sodium chloride solution (pH 7.0).
Was washed by sending 6.0 L of 0.01 g of phosphate buffer and 0.17 M sodium chloride solution (pH 7.0).
(0) was fed in 3.0 liters to elute AT-III. To further purify the eluted fraction, heparin-sepharose gel treatment was performed again. That is, a column packed with about 370 ml of a heparin-Sepharose gel pre-equilibrated with a 0.02 M phosphate buffer, pH 7.3,
-The eluate of the Sepharose FF gel was loaded at 5-10 ° C. Subsequently, under the same temperature conditions, a 0.02 M phosphate buffer solution and a 0.3 M sodium chloride solution (pH 7.3) were used.
After washing with 3.0 liters, each was washed with 0.02M phosphate buffer and 2.0M sodium chloride solution (pH 7.3).
AT-III was eluted at 0 liter.

Next, the solution eluted from the heparin-Sepharose gel was concentrated and buffer exchanged at 10 ° C. or lower using the same ultrafiltration apparatus as described above. As an exchange buffer, a 0.02 M citrate buffer and a 0.07 M sodium chloride solution (pH 7.0) were used and diluted and adjusted to 10 at an absorbance of 280 nm. Then A
The T-III solution was freeze-dried. The obtained dried product was subjected to a heat treatment at a product temperature of 66 ± 1 ° C. for 96 hours. After dissolving this powder in water for injection, 10 mg of sodium L-glutamate
/ Ml, and a virus removal membrane (Asahi Kasei,
(Planova 35N) treatment and freeze-drying to obtain a purified AT-III preparation A of the present invention.

Test Example 1 1) Comparative test between preparation A of the present invention and commercially available preparations B to E A comparison test between AT-III preparation A of the present invention and commercially available AT-III preparations BE was performed. All commercially available AT-III preparations are AT
It is obtained by inactivating the virus by heating the aqueous solution containing -III. The test items compared were AT-III activity and protein content. The following test materials and test methods,
The test results will be described in order. 2) Test Materials and Test Methods Sample Solutions Formulations A to E were filled in vials and freeze-dried, and dissolved in their respective dissolution solutions to prepare sample solutions A to E. Measurement of AT-III activity The AT-III activity was measured according to the AT-III titration method described in the standards for biological preparations. Quantification of protein content The assay was performed using a Bio-Rad Protein Assay Kit (manufactured by Bio-Rad) using bovine serum albumin (manufactured by Bio-Rad) as a standard protein.

3) Test results AT-III activity, protein content and specific activity Measurement results of AT-III activity and protein content and specific activity are shown in [Table 1].

[Table 1] As is evident from Table 1, the specific activity of the preparation A of the present invention was as high as 5.6 IU / mg, whereas the commercially available preparations BE were 4.5 to 5.3 IU / mg. mg.

Test Example 2 1) Preparation A of the present invention using a heparin sepharose column
Analysis of Commercial Formulations B to E Using a heparin sepharose column, the formulation A of the present invention and the commercial formulations BE were analyzed. First, sample solutions A to E prepared from preparations A to E were loaded on a column of heparin sepharose (manufactured by Amersham Pharmacia), and AT-III was added.
Were compared. Furthermore, the peak fraction of each protein eluted from the heparin Sepharose column for the preparation A of the present invention and the commercially available preparations B to E was fractionated, and the fractionated fraction was subjected to SDS polyacrylamide gel electrophoresis.
Analysis by Western blot and N-terminal partial amino acid sequence analysis were performed. Hereinafter, the test materials, test methods, and test results will be described in this order.

2) Test Materials and Test Methods Sample Solutions Formulations A to E were filled into vials, lyophilized, and dissolved in respective dissolving solutions to prepare sample solutions A to E. All sample solutions were measured for AT-III activity using the AT-III titration method described in the standards for biological preparations, and adjusted to a concentration of 18.7 IU / ml based on the AT-III activity. Heparin Sepharose column was loaded. Measurement of AT-III activity The activity of the AT-III fraction eluted from the heparin Sepharose column was measured using a test team AT-III2 kit (Daiichi Kagaku). Heparin Sepharose Chromatography 16.7 ml of a sample solution whose AT-III activity concentration was adjusted was loaded on a heparin Sepharose 6FF (manufactured by Amersham Pharmacia) column equilibrated with 0.02 M Tris-HCl buffer (pH 7.4). AT-III was eluted using a linear concentration gradient of 0.02 M Tris-HCl buffer, 0-2.0 M sodium chloride solution (pH 7.4). The peak was detected by measuring the absorbance at 280 nm. SDS polyacrylamide gel electrophoresis The gel concentration was adjusted to 7.5%, and the method of Laemmli [Natur
e, Vol. 227, 680 (1970)]. Western blot After SDS polyacrylamide gel electrophoresis, transfer to nitrocellulose membrane (Amersham Pharmacia)
The test was performed using a P (Horse Radish Peroxidase) -labeled anti-AT-III polyclonal antibody (manufactured by Cedarlane).

3) Test results AT-II of each sample solution from a heparin sepharose column
I. Elution Pattern Comparison The elution patterns of the sample solutions A to E prepared from the preparations A to E from the heparin sepharose column are shown in FIG. 1 to FIG. As shown in each figure, elution of the main peak was observed around 1.2 to 1.3 M sodium chloride concentration in all the sample solutions. In each figure, as is clear from the OD 280 nm elution pattern indicated by the dotted line, in all the samples, elution of the low heparin affinity protein was observed in the elution fraction having a sodium chloride concentration of 1.0 M or less. The sample liquid A was the least. SDS polyacrylamide gel electrophoresis analysis of the peak fraction eluted from the Heparin Sepharose column of sample liquids A to E Each of the elution peak fractions from the Heparin Sepharose column of sample liquids A to E was subjected to SDS polyacrylamide gel electrophoresis, The images are shown in FIG. 6 to FIG. No. of each figure. Lane No. in the electrophoresis image. And fraction No. Is shown in [Table 2].

[0016]

[Table 2] As is clear from FIGS. 6 to 10, sample liquids A to
In any of E, a band showing the same mobility as the band of the main peak was observed in the peak fraction eluted with a low sodium chloride concentration. In sample solution A, bands other than the band showing the same mobility as the main peak were not observed, but in sample solutions B to E, the elution peak fractions having a sodium chloride concentration of 0.3 to 0.7 were all present. , A band having a mobility lower than the mobility of the main peak was observed. All of the components of these bands reacted with the anti-AT-III antibody in the Western blot.

The components of the bands obtained from the sample solutions B and C were subjected to N-terminal partial amino acid sequence analysis, respectively.
In each case, two types of N-terminal amino acid sequences were detected. That is, one fragment has the same amino acid sequence as the N-terminal amino acid sequence of human AT-III (SEQ ID NO: 1) (SEQ ID NO: 2).
And 3) and another is Ser39 of human AT-III.
4 (the fourth amino acid Ser in SEQ ID NO: 4 is
It corresponds to amino acid Ser at position 394 of human AT-III. )
Fragments (SEQ ID NOS: 5 and 6) having the same sequence as the sequence from the above were detected. From this result, it is considered that the component of this band is that AT-III is cleaved at least at the reaction site (Arg393-Ser394). From the above results, the preparation A of the present invention contained little AT-III with low heparin affinity, and no cleaved AT-III was observed. Therefore, the preparation of the present invention contained cleaved AT-III. A high purity AT-III preparation was revealed.
In contrast, all of the commercially available AT-III preparations B to E contain AT-III with low heparin affinity, and A
T-III was confirmed.

[0018]

The purified AT-III of the present invention is cleaved AT-III
Is substantially not contained, so that a highly safe preparation can be provided. That is, according to the production method of the present invention, despite having a virus inactivating effect at least as high as the virus inactivating effect obtained by the conventional method, denaturing or inactive AT-haparin with low heparin affinity. III
, And in particular, a purified AT-III substantially free of cleaved AT-III is obtained, so that an additional step for removing them is unnecessary. In addition, since this method is suitable for mass production, it is extremely useful as an industrial production method.

[Sequence list] SEQ ID NO: 1 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence His-Gly-Ser-Pro-Val-Asp-Ile-Cys-Thr-Ala- Lys-Pro-Arg-Asp-Ile-Pro-1 5 10 15 Met-Asn-Pro-Met 20 20 SEQ ID NO: 2 Sequence length: 10 Sequence type: amino acid Topology: Linear Sequence type: Peptide Xaa: unknown sequence His-Gly-Ser-Pro-Val-Asp-Ile-Xaa-Thr-Ala 10 1510 SEQ ID NO: 3 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type : Peptide Xaa: unknown sequence His-Gly-Xaa-Pro-Val-Asp-Ile-Xaa-Thr-Xaa-Xaa-Pro 121510 SEQ ID NO: 4 Sequence length: 20 Sequence type: amino acid Topology: Type of linear sequence: Peptide sequence Ala-Gly-Arg-Ser-Leu-Asn-Pro-Asn-Arg-Val-Thr-Phe-Lys-Ala-Asn-Arg-1 5 10 15 Pro-Phe-Leu -Val 20 20 SEQ ID NO: 5 Sequence length: 10 Sequence type: amino acid Topology: linear Type: Peptide Sequence Ser-Leu-Asn-Pro-Asn-Arg-Val-Thr-Phe-Lys 10 1 5 10 SEQ ID NO: 6 Sequence Length: 12 Sequence Type: Amino Topology: Linear Sequence Type: Peptide Xaa: Unknown sequence Ser-Leu-Asn-Pro-Asn-Arg-Val-Xaa-Phe-Xaa-Xaa-Asn 12 1 5 10

[Brief description of the drawings]

FIG. 1 Heparin Sepharose column elution pattern of Formulation A.

FIG. 2 Heparin Sepharose column elution pattern of Formulation B.

FIG. 3 Heparin Sepharose column elution pattern of Formulation C.

FIG. 4 Heparin Sepharose column elution pattern of Formulation D.

FIG. 5 Heparin Sepharose column elution pattern of Formulation E.

FIG. 6 is an SDS polyacrylamide gel electrophoresis image of the elution peak of formulation A.

FIG. 7 is an SDS polyacrylamide gel electrophoresis image of the elution peak of Formulation B.

FIG. 8 is an SDS polyacrylamide gel electrophoresis image of the elution peak of formulation C.

FIG. 9 is an SDS polyacrylamide gel electrophoresis image of the elution peak of Formulation D.

FIG. 10 is an SDS polyacrylamide gel electrophoresis image of the elution peak of Formulation E.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Hideya Maruyama 3-1, Shinizumi, Narita-shi, Chiba Japan Pharmaceutical Co., Ltd. Narita Plant (72) Inventor Masataka Tanabe 3-1, Shinizumi, Narita-shi, Chiba 1 Nihon Pharmaceutical Inside the Narita Plant Co., Ltd. (72) Inventor Kenji Kaneko 3-1, Shinizumi, Narita City, Chiba Prefecture Nippon Pharmaceutical Co., Ltd. Narita Plant F-term (reference) 4C084 AA06 AA07 BA44 DC35 MA17 MA23 MA44 MA66 NA01 NA06 ZA542 4H045 AA10 AA20 BA53 CA42 EA24 GA10 GA23 GA26 HA06

Claims (4)

[Claims] 1. A purified antithrombin-III substantially free of active virus and cleaved antithrombin-III. 2. The method of claim 1, wherein the human plasma or the induced human plasma fraction is at 13 ° C.
Subjected to a purification step using immobilized heparin at a temperature not exceeding, and further subjected to a drying step, and then heating the resulting dried antithrombin-III until the virus is inactivated. A method for producing purified antithrombin-III substantially free of cleaved antithrombin-III. 3. After the purification step using immobilized heparin and before the drying step, the solution containing antithrombin-III is absorbed and desorbed by a strong anion exchanger at a temperature not exceeding 13 ° C. and fixed. 3. The method for producing purified antithrombin-III according to claim 2, which is subjected to a purification step using heparin chloride. 4. The method according to claim 4, wherein the inactivation of the virus is carried out at 50 to 80.degree.
The method for producing purified antithrombin-III according to claim 2, which is carried out for up to 120 hours.