RU2583931C2 - Method of producing thrombin concentrate - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, specifically to a method of extracting a purified thrombin concentrate. Method for preparing thrombin concentrate, free from viruses comprising cryofractionation of fresh frozen human blood plasma, prothrombin complex separation on an anion exchange sorbent DEAE Sepharose Fast Flow, activation of prothrombin solution of CaCl₂ prothrombin complex composed of thrombin to yield viral inactivation by solvent-detergent and purified thrombin prepared cation exchange sorbent Fractogel EMD-SO₃ followed by sterile filtration and lyophilisation.

EFFECT: described method increases purity and output of end product.

1 cl, 1 tbl, 1 ex

Description

The invention relates to the field of pharmaceutical industry, namely to biotechnology for producing hemostatic drugs.

Thrombin is a natural component of the blood coagulation system, which is formed in the body from an inactive form of the enzyme, prothrombin, with the enzymatic activation of the latter by thromboplastin.

Industrial production of thrombin preparations from blood plasma is associated with its clinical use as the main hemostatic. In therapy, thrombin is used as an independent tool or more often together with fibrinogen in the form of fibrin glue.

Fibrin glue is a highly effective local hemostatic agent used in modern surgery. The main components of the adhesive are fibrinogen, coagulation factor XIII and thrombin are released from the donor plasma. When applied to the wound surface, the fibrin glue polymerizes with the formation of an elastic fibrin film of white color. This process repeats the main stages of the physiological process of blood coagulation and allows you to stop diffuse bleeding, glue and fix tissue, as well as accelerate wound healing.

The preparations of purified thrombin are produced both from the plasma of human donated blood and by genetic engineering methods.

As the initial stage in the production of preparations derived from donated plasma, including thrombin-containing ones, the cryofraction method of freshly frozen donated blood plasma is usually used, followed usually by several stages of chromatographic fractionation. The raw material for thrombin concentrate is usually the so-called prothrombin complex concentrate (PCC), obtained from cryosupernatant (CHF) of donor blood plasma. RCC includes vitamin K-dependent coagulation factors: FII (prothrombin), FVII, FIX, FX, proteins C and S, etc.

PPSB is isolated from SPM by chromatographic fractionation or precipitation. The chromatographic fractionation method, developed in the 1960s and the most common at the present time, consists in the interaction of SPE with a weak anion-exchange sorbent.

Since thrombin is in plasma in an inactive form, in the form of prothrombin, the next stage of preparation after primary purification is the activation of FII-containing plasma fractions.

There are several ways to activate. The first method is the activation of prothrombin as part of PCC with thromboplastin, usually in the presence of calcium chloride. This lipoprotein activates factor VII, which in turn activates factor X, which converts prothrombin to thrombin. The disadvantage of this method is that thromboplastin is usually obtained by rough cleaning from homogenized brain, lungs and intestinal tissues. This method cannot be used for the production of human thrombin, since the quality of the resulting product is very dependent on raw materials, which have a high risk of cross viral contamination.

The second activation method is to use the components of cobra venom. However, it was found that cleavage by some types of poisons occurs at positions inside the prothrombin, different from the natural activator - factor Xa. Therefore, the use of this method is also dangerous and inapplicable for the production of drugs for clinical use.

The third method involves activation under conditions close to physiological: prothrombin is activated in the presence of factors IX, X, VII, phospholipids and calcium ions. The thrombin thus obtained is often unstable and requires the addition of stabilizing proteins, polyols and / or sugars.

After activation, the thrombin-containing solution is subjected to final purification. In addition to common fractionation methods on ion-exchange and gel-filtration sorbents, thrombin can also be purified using heparin, benzamidine-affinity sorbents, as well as by chromatography of hydrophobic interactions, using a sorbent with attached phenyl groups. In addition, there are works describing the use of metal-chelate chromatography with immobilized metal ions for the purification of thrombin.

Since human thrombin is obtained from donated plasma, there is a risk of thrombin contamination by various viruses. Therefore, all thrombin preparations intended for clinical use should be subjected to viral inactivation. For thrombin, this is usually a solvent-detergent treatment and / or pasteurization and sterilizing filtration.

Table 1 shows examples of methods for producing thrombin that do not use components of biological origin for activation.

A method for producing thrombin with activation on a sorbent has also been proposed. In this method, PCC purified on DEAE-SepharoseCL6B was applied to a DEAE-Sepharose or Fractogel EMDSO ₃ sorbent in a thermostatic chromatographic column and filled with a buffer solution containing CaCl ₂ . Activation occurred in a closed column at 20 ° C for 64 hours. After that, activated thrombin was eluted with the start buffer, while prothrombin, which was not subjected to activation, remained adsorbed on the sorbent. The purity of the obtained thrombin on both sorbents was> 2000 IU / mg.

The aim of the present invention is to develop a method for the industrial production of a stable preparation of highly purified thrombin.

The development of technology for the production of highly purified thrombin was carried out in laboratory conditions, followed by optimization of the selected technique, and then scaled up in pilot production.

The raw material for thrombin production is freshly frozen plasma of donated blood.

The process of obtaining thrombin from plasma of donated blood includes the following stages:

1. cryofractionation of FFP;

2. isolation of the prothrombin complex on an anion-exchange sorbent;

3. activation of prothrombin as part of the prothrombin complex to produce thrombin;

4. viral inactivation by solvent-detergent method;

5. purification of the obtained thrombin on a cation exchange sorbent.

At the stage of cryofractionation, FFP is divided into 2 fractions by the method of controlled thawing and centrifugation: cryoprecipitate (KP) and cryosupernatant (KSN). Detachable KP is used as raw material to obtain FVIII concentrates and fibrinogen.

After adjusting the pH and ionic strength, the prothrombin complex concentrate (PPSB) is isolated from the obtained SPH.

As an anion-exchange sorbent in the PPSB separation step, it is possible to use weak anion exchangers containing DEAE groups, preferably sorbents based on modified agarose, preferably DEAE-Sepharose Fast Flow sorbent.

PPSB is isolated using Tris citrate buffer solutions with a pH of 7.8 in a stepwise gradient of ionic strength created by increasing the concentration of NaCl in the starting buffer solution. The linear speed of the process is 80-90 cm / h.

The yield of prothrombin in the PPSB fraction is at least 90%.

PPSB activation is carried out under conditions close to physiological. The activator of prothrombin is a solution of CaCl ₂ . The concentration of calcium ions of Ca ^{2+ is} calculated taking into account the amount of citrate in the buffer solution so that the concentration of free calcium ions of Ca ²⁺ is 2-20 mmol / L. The pH of the solution is maintained at 7.0. The activation process lasts about 24 hours at a temperature of 23 ° C. The thrombin yield is at least 90%.

The obtained thrombin solution was subjected to viral inactivation by SD-processing using a 0.3% solution of tri-n-butylphosphate (TnBP) and a 1% solution of Triton X-100 for 6 hours at room temperature at a pH value of 6 before the start of the chromatographic purification step. 5 to 7.8.

The virus-inactivated thrombin solution is purified by cation exchange chromatography, preferably using a Fractogel EMD-SO ₃ ion exchanger. Chromatographic purification is carried out using Tris-HCl buffer solutions with pH from 6.5 to 7.8 in a stepwise gradient of ionic strength created by increasing the concentration of NaCl in the starting buffer solution. The linear speed of the process is 80-90 cm / h.

The target fraction of purified thrombin has a specific activity of at least 2000 IU / mg of total protein.

The thrombin yield at the chromatographic purification step is at least 90%.

Human albumin is added to the resulting thrombin solution as a stabilizer, filtered sterilely, poured into vials and lyophilized.

The total yield of thrombin from 1 liter of FFP is at least 90%.

The following is a specific embodiment of the invention.

Example 1

1. 30 l of freshly frozen donor plasma with a prothrombin activity of 0.7-1ME / ml * was thawed in a reactor at a temperature of -0.5 ° C and cryosupernatant (KCH) was isolated by centrifugation in a flow centrifuge at a speed of 18,000 rpm and 3 ° C. The loss of prothrombin was less than 10%.

2. Chromatographic fractionation of 30 L of SPF was carried out on a column with a diameter of 200 mm filled with a DEAE Sepharose Fast Flow sorbent using tris citrate buffer solutions of pH 7.2 by increasing the concentration of sodium chloride in the starting buffer solution from 0.08 M to 0.22 M. Losses of prothrombin at the stage of application of SPE and washing with the starting buffer solution did not exceed 1% of the initial content. The fraction containing prothrombin was eluted in a volume of about 1 L with a buffer solution containing 0.22 M sodium chloride. The prothrombin yield was at least 90%.

3. Thrombin activation was carried out at room temperature with stirring using CaCl ₂ as an activating agent. The activation raw material is the fraction obtained at the previous stage containing prothrombin with an activity of about 20 IU / ml *, to which calcium chloride was added in the form of a 0.5 M solution to a concentration of free Ca ^{2+ ions} of 8 mmol / L. The activation time was 24 hours. After activation, the resulting mixture was centrifuged for 10-15 minutes at 4000 rpm. Under these conditions, about 90-95% of prothrombin is transformed into thrombin.

4. Viral inactivation of a thrombin solution was carried out by the solvent-detergent method, adding Triton X-100 to a final concentration of 1% and tri-n-butyl phosphate to a final concentration of 0.3% at pH 6.8.

5. Chromatographic purification of the thrombin-containing fraction was carried out on a column filled with Fractogel EMD-SO ₃ ion exchange sorbent using Tris-HCl buffer solutions, pH 6.5, by increasing the concentration of sodium ions in the starting buffer solution. The concentration of sodium chloride in the starting solution was 100 mmol / L. After application of the sample, washing was carried out with 10 volumes of starting buffer solution to remove unbound proteins and reagents used for viral inactivation. Next, the sorbent was washed with buffer solutions with intermediate ionic strength. At this stage, thrombin losses did not exceed 1% of the initial amount. Thrombin was eluted by increasing the concentration of sodium ions to 1000 mmol / L. The yield of thrombin * at the stage of chromatographic purification was at least 90%. The specific activity of purified thrombin exceeded 2000 IU / mg protein **.

6. Human albumin is added to the resulting thrombin solution as a stabilizer, sterilely filtered, poured into vials and lyophilized.

* Determination of prothrombin time was carried out using thromboplastin-calcium reagent and substrate-deficient plasma by prothrombin

** Thrombin concentration was determined coagulometrically by clotting time when a standard fibrinogen solution was added to the sample.

*** The concentration of total protein was determined by the Bradford method.

Claims (1)

A method for isolating a purified thrombin concentrate, freed from viruses, characterized by a high yield, purity and high stability, which consists in cryofractionation of the feedstock represented by freshly frozen human donor blood plasma, isolation of the prothrombin complex on an anion exchange sorbent DEAE Sepharose Fast Flow, prothrombin activation with CaCl ₂ solution in the composition prothrombin complex to obtain thrombin, viral inactivation by solvent-detergent method and purification of the obtained thrombin on cation Fractogel EMD-SO ₃ exchange sorbent, characterized in that the amount of CaCl ₂ solution acting as a prothrombin activator is calculated taking into account the interaction of CaCl ₂ with sodium citrate, which is part of the buffer used in the isolation of the prothrombin complex on an anion exchange sorbent, so that the concentration of free Ca ^{2+ ions} in the solution at the activation stage is from 2 to 20 mmol / L.