FI75057C - FOERFARANDE FOER FRAMSTAELLNING AV ETT FAKTOR-XA-PREPARAT. - Google Patents

Description

! 75057

Method for preparing a factor Xa preparation f

Divided separated from application 783880 (patent 64 466) -Avdelad frän trap 783880 (patent 64 466)

The invention relates to a process for the preparation of a factor Xa preparation. Factor Xa can be utilized in the determination of prothrombin in a biological agent such as blood fluid.

Determination of prothrombin levels is an important clinical parameter in monitoring the progress of anticoagulant therapy. Likewise, there is an important indication of factor II deficiency, which may be congenital as well as acquired, i.e., a consequence of the primary underlying disease.

Since the development of prothrombin assays from the native thrombin substrate fibrinogen, the use of synthetic dye-generating substrates, e.g., peptide-β-anide derivatives, has shifted. Tissue thromboplastin was used as an activator. The disadvantages of this method are, for example, that the best possible activation time varies depending on the prothrombin content and the lack of specificity. When other substrates were used, Akti may have a lower blood fluid concentration for the determination of prothrombin in normal blood fluid. However, by diluting the blood fluid so strongly, it was not possible to achieve complete activation in blood fluids with low prothrombin activity. Staphylocoagulase α and snake poisons were also used as activators. They activate prothrombin immediately.

However, it has been shown that said toxins also at least partially activate the prothrombin PIVKA, which is formed during treatment with orally administered anticoagulants. Stafy locoagulase similarly alters this so-called Pivka-prothrombin.

FI patent specification 64,466 (parent application 783880) describes a method and reagent for the determination of prothrombin in which factor Xa, preferably human factor Xa, is used as the prothrombin activator.

2 75057

The above method and reagent make it possible to perform an experiment with fewer sources of error and with higher accuracy and sensitivity as well as better reproducibility. This assay is specific, i.e., it detects only prothrombin1 but not PIVKA prothromb11. In this case, all protrombllnl changes rapidly and completely to the thrombin, without the necessary activator in any form taking part in the color reaction.

The principle of the above assay can be explained as follows: thrombin cleaves, e.g., o 1 gopeptide in which the carboxyl group of arg1n11n1n is attached to the carboxyl group p-n1troanl111nl as an amide bond as a color-generating group, p-nltroan111nn:

n-Tos-Gly-Pro-Arg-pNA 1 1 # n ^> N-Tos-Gly-Pro-Arg-OH + pNA

2 °

N-Tos-Gly-Pro-Arg-pNA and N-Cbz-Gly-Pro-Arg-pNA (Chro-mozym TH, Boehringer Mannheim GmbH), as well as HD-Phe-P1p-Arg-pNA, have proven to be particularly suitable substrates for thromblin. (S-2238, Kabi) and Bz-Phe-Val-Arg-pNA (S-2160, Kabi). The yellow color of free p-nitroan11 can be measured photometrically at wavelengths of about 390-410 nm, with the amount of color released per unit time being proportional to the enzyme activity. The buffers used are suppository trls and / or imidazole buffers with a pH of about 8-9, optionally with the addition of hydrochloric acid and / or sodium chloride.

Phospholipids and calcium chloride are used as co-reagents to activate prothromblin.

Substrate addition occurs after prothrombin is completely converted to thrombin in the test solution, after the addition of factor Xa.

The process for the preparation of factor Xa or a compound according to the invention is characterized in that the blood fluid, suitable human blood fluid, is treated with a prothromblin activator, after centrifugation a protein adsorbent 75057 is added, the precipitate is eluted with protein X possibly some soluble cesium salt. It is preferred to use Echis-carinatus venom as the prothrin activator, but snake poisons such as Taipan snake venom (Oxyuranus scutel1atus) and trypsin are also suitable.

The coagulate obtained after treatment with prothrombin activator 11a, which is separated by centrifugation, consists in part of fibrinogen, anti-thrombin and thrombin.

Barium sulphate, citrate and oxalate are the most suitable adsorbents for the protein, but also aluminum hydroxide, DEAE-Sephadex N-diethyl-2- (2-hydroxy-1-propyl) ammonium (modified from dextran gel). For the elution of the protein, for example, aqueous trisodium citrate solution, phosphate buffer or sodium chloride solution can be used. The actor of factor X Akti is Russell's viper-venom most suitable. As the soluble cesium salt, cesium chloride is most preferred. In the case that ingredients which adversely affect the coagulation system, for example calcium-binding components such as citrate ions, are present in the eluate after treatment with the protein elution 1a, when the protein elution is sodium citrate, it is necessary to increase the diary. In said case, the dialysis is performed, e.g., against buffered physiological saline solution (Michael 1 s buffer a) at low temperature, i.e. at about 4 ° C. After treatment with factor X activator and addition of a solution of calcium monopoly, the preparation is left to stand at low temperature, preferably at 4 ° C, before use, until factor X is completely converted to factor Xa. In addition, other steps can be performed to obtain highly pure factor Xa. These 1-purification steps may involve gel filtration with a molecular sieve separating the range of 50,000 to 100,000, e.g., using ____ to τΓΖ. ----— 4,75057 Sephadex G 100 or Biogel P 100 to elute sodium acetate buffer 11a (0.4 m, pH 7). Ammonium sulphate precipitation (e.g. 45-55% solution, pH 6-8) and ion exchange chromatography using DEAE-Sephadex A 50, DEAE-sel1 or QAE-Sephadex / sii are also possible, in which case Na / K-phosphate can be used as buffer. (e.g. 0.02 m, pH 6.8) or a gradient of 0.1-1.0 m NaCl. Hydroxyapatite treatment (phosphate buffer 0.2-0.5 m, pH 6.8) as well as preparative electrophoresis and possibly ultrafiltration are also possible. It is preferred to combine said methods. Thus, highly purified factor Xa can be used in a prothrombin assay in the form of a solution dissolved in e.g. veronal or Michaelis buffer, physiological saline or assay buffer. However, in the prothrombin assay, this purification lie can be omitted and a manufacturer-Xa preparation enriched as described above can be used.

The factor Xa preparation according to the invention can be used in a prothrombin assay reagent which, in addition to the factor Xa agent, contains trls and / or imidazole buffer α, in which case phospholipids from human brain and calcium chloride dia and a synthetic thrombin substrate are preferably used as co-reagents. Most preferably, such a reagent contains 0.3-6.5 μg / ml phospholipids, 0.7-10 mM / l calcium chloride di a, and 150-380 μm / l substrate with a factor Xa concentration corresponding to 0.2-2.5. % of blood fluid extract. The reagent may be in either dry or dissolved form.

The method for determining prothrombin using the factor Xa preparation prepared according to the invention is specific because factor Xa affects only normal prothrombin and not PIVKA prothrombin. The Akti watters used so far do not specifically have a specific effect. The addition of a standardized factor Xa agent ensures that a sufficient amount of activator is present in each case. It was very surprising that after the addition of factor Xa, all of the prothrombi1n is rapidly and completely converted to thrombi1n, without what is very essential itself in any way taking part in the color reaction. Namely, it is known that factor Xa has a direct degradative effect on e.g. synthetic peptide -d-ni troani 1 i di- derivatives, such as the derivative Chromozym TH. It was also particularly surprising that human factor Xa is more advantageous to use than e.g. bovine factor Xa. It has proved very advantageous to add factor Xa in a certain amount. Activation by factor Xa in the blood fluid itself would then lead to unsatisfactory results. Furthermore, it was surprising that an activator as simple as the process according to the invention can provide an activator which guarantees performance, in particular technical applicability, and which, due to its origin, namely human blood fluid, can generally be sufficiently produced.

The following Example 1 illustrates a method of the invention for the preparation of ice Xa and Example 2 illustrates the use of a factor Xa preparation according to the invention for the determination of prothrombin.

Example 1

Preparation of factor Xa or preparation

Blood from healthy blood donors with an average age of 30 years, half of whom are women and half men, is used to produce normal blood fluid. The blood fluid containing 25 mM sodium citrate is subjected to a second centrifugation at 20,000 g for 30 minutes at 4 ° C after 30 minutes at room temperature. For 4 ml of normal blood fluid, add 1 ml of a 1/30 m aqueous solution of calcium chloride and 2 drops of Echis-carinatus-venom (Sigma V 8250; stock solutions of 1 mg / ml water each time), and the mixture is kept under 6 75057 o for about 2 hours in a water bath (37 ° C). The formed coagulate is separated and 150 mg of barium sulfate is added to the residual fluid obtained from each of 4 ml of normal blood fluid. The mixture is stirred for 30 minutes at room temperature, after which it is centrifuged. The separated liquid is removed and the precipitate is washed four times, each time with about 4 ml of physiological saline solution. The centrifugation product is eluted with 2 ml of 0.2 M trisodium citrate solution (pH 7.0). After re-centrifugation, the separated liquid is dialyzed against physiological saline at 4 ° C.

The dialysis product is stored in small doses of about 250 [mu] l in mouth / rye at -20 [deg.] C. Per dose, 30 [mu] l of 0.1 m potassium chlorine dl 11 solution and 1 [mu] l of Russell's vlper-venom'1a are added at room temperature. , Wellcome), and the dose is allowed to stand for about 14 hours at 4 C. This preparation is available for the experiment (2 μl corresponding to 1% blood fluid extract). It can be lyophilized. Further, cleaning can be performed, as described in detail on page 4.

Example 2

Method for the determination of prothromblin and thromblin Prepare test mixtures containing

Substance Volume The rest was brought orally

Buffer solution 400, minus other added amounts

Test blood 1.5 μl 0.25-1.25%

Phospholop1dit 2 μl 1.25 μg / ml

Potassium chloride di-40 μl (0.1 m) 10 mM / l solution

Activator (made of 2 μl corresponding to 1% ice-Xa or miste precursor liquid fraction according to label 1)

II

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The buffer solution is prepared as follows:

Stock solution A: 0,1 M / l tris- + 0,1 M / l imidazole buffer + 0,1 M / l hydrochloric acid

Stock solution B: 0.1 M / l tris- + 0.1 M / l imidazole buffer + 0.1 M / l sodium chloride.a

Stock solution C: 0.2 M / l sodium chloride Working buffer (pH = 8.4) is prepared by combining stock solutions in a volume ratio of A: B: C = 1: 1: 2. The phospholipids used are acetone / ether extract obtained from the human brain according to Bell and Alton.

The components of the test mixture are pipetted at 37 ° C in the order indicated directly into the microcuvette of the spectrophotometer "Aminco DW 2" with a layer thickness of 1 cm. The incubation time for factor Xa is 120 seconds. Then 50 [mu] l (1.5 mM / l) of substrate (Chromozym TH, Boehringer Mannheim GmbH or Substrat 2238, Kabi) is added, with a final concentration of 187.5 [mu] M / l (Chromozym TH) or 148 [mu] M / l, respectively ( S 2238). After a thorough agitation as soon as possible, the measurement immediately follows.

Absorption over time is described directly at the pointer travel rate in minutes per centimeter in the horizontal plane and absorption in millimeters per millimeter of the full scale in the vertical plane. In this case, different absorption sensitivities and flow rates can be selected. Thus, the change in absorption per minute can be calculated from the following formula: measured absorption in millimeters - x absorption sensitivity full scale in millimeters pointer speed in min / cm x measuring range

Example with a measuring range of 10 cm, a vertical reading of 43 mm, a full scale of 252 mm, an absorption sensitivity of 0.5 and a travel speed of 0.033: 43 ^ c I -I— ^ Q k 252 '„change in absorption - = 0.258 --- 0.033x 10 min / cm minute

Claims (8)

75057 A process for the preparation of a factor Xa preparation, characterized in that a blood fluid, preferably a human blood fluid, is treated with a prothrombin activator, after centrifugation a protein adsorbent is added, the precipitate is eluted with a protein eluent and a factor X activator and possibly a soluble calcium salt are added to the eluate. . Process according to Claim 1, characterized in that the prothrombin activator is Echis-carinatus-venom. Process according to Claim 1 or 2, characterized in that the protein adsorbent is barium sulphate. Process according to one of Claims 1 to 3, characterized in that the protein is eluted with an aqueous solution of sodium citrate. Process according to one of Claims 1 to 4, characterized in that the activator of factor X is Russell viper-venom. Process according to one of Claims 1 to 5, characterized in that the soluble calcium salt is calcium chloride. Process according to one of Claims 1 to 6, characterized in that, in the case where the elution is carried out with protein eluents containing calcium-binding components, such as citrate and oxalate, the eluate is dialyzed. Process according to one of Claims 1 to 7, characterized in that the further purification of the obtained factor Xa preparation is carried out using gel filtration and / or ammonium sulphate precipitation and / or ion-exchange chromatography and / or preparative electrophoresis and / or ultra-centrifugation. Il 75057