Method for enriching donor blood plasma cryoprecipitate with factor viii

FIELD: medicine.

SUBSTANCE: invention relates to methods for preparing biologically active substances from donor blood. Method for enriching donor blood plasma cryoprecipitate with factor VIII involves defrosting freshly frozen donor blood plasma at +1-4°C to obtain cryofractions from plasma cryosupernatant and cryoprecipitate. After formation of the donor blood freshly frozen plasma dry calcium chloride is added in the amount from 0.001 to 0.004 mole/l followed by addition of sodium citrate in the amount from 0.05 to 0.10 mole/l and stirring at +1-4°C for 10-15 min. Then prepared cryoprecipitate deposit is separated by centrifugation. For preparing cryoprecipitate freshly frozen plasma is used prepared by usual method with using conventional preserving agents. Using the method provides significant increasing the content of factor VIII in cryoprecipitate being without appreciable co-precipitation of inert proteins.

EFFECT: improved enriching method.

2 tbl, 3 ex

 

The invention relates to medicine and concerns cryoprecipitate plasma donor blood and method of its production.

FVIII is one of the most labile factors system gemokoagulyatsii. The concentration of FVIII in the plasma does not exceed 1-2×10-5%.

FVIII is very sensitive to proteolytic degradation, and its concentrates, isolated from plasma, it is possible to detect partially degraded, but the active form with a molecular weight of from 280 to 170 kDa.

Deficiency of FVIII causes disturbances in the coagulation of the blood, observed in severe hereditary disease - hemophilia A.

For the treatment of hemophilia And uses drugs on the basis of FVIII concentrates.

Raw materials for the industrial allocation of FVIII concentrates is cryoprecipitate (CP) plasma donor blood, which is a fraction of plasma of blood, which in addition to FVIII include fibrinogen, fibronectin, von Willebrand factor system gemokoagulyatsii and other proteins [1]. KP is obtained from polerowanie fresh frozen plasma (FFP) by thawing at a temperature of from 1° to +2°C. This process is called cryoprecipitate.

The main drawback of all existing technologies for antihemophilic preparations of factor VIII is the low yield of FVIII, not exceeding 200-250 IU (international units) of FVIII 1 LSSP, the content of FVIII which is not less than 800 or 900 IU/l [2].

This is primarily due to the low content of FVIII in KP obtained at the initial stage of cryoprecipitate NWS in industrial technological processes of obtaining drugs on the basis of FVIII. On average, the content of FVIII in KP is about 30.0-35.0% of its content in the source plasma [2].

In the patent [3] was first proposed to modify the process cryofracture by adding in containers with plasma carboxylic acids and their salts before freezing containers. Stage thawing FFP thus carried out without changes.

It was shown that adding to the plasma donor blood before freezing of sodium citrate in an amount of from 2 to 10 weight % leads to a sharp increase in the weight of KP obtained at the stage of cryoprecipitate.

In our proposed method, the thawing process (cryoprecipitate) NWS modified by additives of certain salts, which leads to increased levels of factor FVIII in the resulting KP.

Use as additives to the plasma salts of carboxylic acids, including citric acid, allows us to consider a patent [3] as a prototype. However, a significant difference of our method from the prototype is that of salt, including sodium citrate are added to asporogenous NWS, obtained in the traditional way, after the stage of cryoprecipitate.

Description of the proposed method.

The raw material used fresh frozen plasma blood (NWS), the last control in accordance with the requirements of regulatory and technical documentation and zagotavlivaetsya in accordance with the "Instructions for collecting and preserving blood from 29.05.95,

We used the following reagents both imported and domestic production classification CHC": trinatriytsitrat 2-water and 5.5-water, sodium chloride, citric acid, TRIS, calcium chloride and anhydrous 2-water. In addition, used a special reagents for coagulopathies measurements ("Behring, Germany) for determination of total protein content by the method of Bradford.

Measurement of the specific activity of factor VIII.

The specific activity of factor VIII coagulation system in the international activity units was determined on coagulometer KS-4A firm "Amelung (USA) single-stage coagulopathies method using graphomania. For the amount of international units of FVIII activity (ME FVIII) took the activity corresponding to the activity of 1 ml of normal plasma.

Determining the concentration of total protein.

The protein concentration in the samples ISM is rali by the method of Bradford using spectrophotometer Ultrospec-450" production "LKB", Sweden.

As the reagent used a dye solution of Kumasi brilliant blue G-250. The protein concentration was determined by calibration curve, constructed using the standard sample solution of human albumin.

Description of the process cryofracture.

NWS removed from the plastic containers are crushed and placed in a cell of a thermostat, pre-cooled to +3°C. carry out defrosting in the cell at +1-4°C for 3-4 h until the formation of cryoprecipitate (KP).

After the formation of the CP is added to the dry sample of calcium chloride to obtain the estimated concentrations (0.001-0.004 mol/l). After 5 min after the addition of calcium chloride into the cell with stirring, add the dry addition of sodium citrate to obtain the estimated concentrations (0.05-0.10 mol/l). Stirred for 10-15 min at +1-4°C. the mixture is Then of cryosupernatant (STOs) and KP centrifuged for 15 min at 4000 rpm at 0°C.

The precipitate KP transferred into a plastic container, frozen and stored at -40°C.

Calculate the output KP in g of l AW by the formula:

η=PCP×1000/VAW,

where RCP- weight KP, g; VSW- the volume of FFP, mil

To determine the specific activity of the obtained KP sample KP dissolved in M Tris-HCl, pH 7.0, containing 0-0 M Cal 2at +37°C for 10-15 minutes

The buffer volume calculated by the formula:

V(ml)=9×Parr.

where Rarr.- weight of sample KP, g; V is the volume of buffer solution.

In the resulting solution to determine the specific activity of FVIII and the concentration of total protein.

Count the total number of ME FVIII in the pattern KP by the formula:

∑ME=(AFVIII/100)×VKP

where AFVIIIspecific activity of FVIII, %;

VCP- the volume of solution CP, ml;

Calculate the weight of protein in the solution of the KP by the formula:

Weightb(g)=(Cb/100)×VKP

where Cbthe concentration of total protein, %;

VCP- the volume of solution KP, Jr.

Calculate the specific activity of the obtained KP by the formula:

AUD.(IU/mg)=∑IU/(Weightb×1000).

Expect the release of FVIII from 1 l AW by the formula:

The output of FVIII (IU/l AW)=∑IU×1000/VAW.

Expect the release of FVIII in % of baseline by the formula:

The output of FVIII (%) = Output FVIII (IU/l NWS)/AFVIII×10,

where AFVIIIthe activity of FVIII in the original EWS, %.

The output of the CP, g/l EWS
Table 1
Comparison of results cryofracture in the absence of additives and in our proposed conditions.
The concentration of sodium citrate, wt.%The output of FVIII,AND UD. media., IU/mg
IU/l EWS% of initial content
011-13260-31030-350.25
2.118-20735-82187-920.44

Table 2.

Cryofracture in terms of patent-prototype
The concentration of sodium citrate, wt.%Weight KP obtained from a fixed amount of SZPFVIII, ME/mlA*beats, IU/mg
00.1 g1.20.12
20.3 g1.80.06
50.9 g2.470.03
Approx.*: AndIDcalculated from presented in the patent-prototype data by weight of the obtained KP and FVIII activity.

From the presented data it follows:

1. In terms of patent-prototype adding 2% of citrate leads to increased release of FVIII in KP 1.5 times compared with the control (180%/120%). In our proposed conditions of adding the same amount of citrate in combination with the addition of chloride is Alicia provides increased output of FVIII in the 2.5-2.6 times in comparison with control, what in the prototype cannot be achieved even when the concentration of citrate 5%.

2. Our terms of cryofracture allow selective deposition of FVIII without noticeable coprecipitation of ballast proteins, which was confirmed by weight of the obtained KP.

3. Our terms of cryofracture provide improve the quality of the KP (AUKincreases from 0.33 to 0.44 IU/mg) compared with control and with patent-prototype that facilitates the process of its further fractionation.

In addition, unlike patent-prototype in the proposed method, salt not added to donor plasma before freezing and after thawing FFP. Thus, our proposed method may receive the CP with a high content of FVIII from any NWS obtained in the usual way on the traditional preservatives.

Examples.

Example 1.

Sample: EWS (volume 60 ml).

EWS from the container was placed in a plastic cylinder and was thawed by placing the cylinder in a thermostat at +1°C for approximately 2 hours. Added dry hitch CaCl2×2H2About equal to 0.021, After 5 min in the cell added dry trehzameshchenny sodium citrate in the amount of 1.34, After 15 min the mixture of the SPE and KP was centrifuged at 3300 rpm at 0°C. the precipitate KP has frozen and stored at -40° C.

To determine the specific activity of the obtained KP it was dissolved in the buffer solution of the following composition: M Tris-HCl, M CaCl2, pH 7.0 at +37°C for 10 minutes

The resulting solution volume of 15 ml specific activity of FVIII, a certain one-step method, amounted to AFVIII=298%.

The total number of FVIII in the sample KP, ∑ME=44.7 ME

Output FVIII=44.7/60×1000=745.5 IU/l AW.

Example 2.

Sample: EWS (volume 60 ml).

NWS to grind and were thawed in a cell thermostat at +2°C for 2 hours. After the formation of the CP is added to the dry sample CaCl2equal to 0.017, After 5 min in the cell added dry trehzameshchenny sodium citrate in the amount of 1.23 g under stirring. After 15 min the mixture of the SPE and KP was centrifuged for 25 min at 3300 rpm

The obtained KP dissolved in the buffer solution of the following composition: M Tris-HCl, M CaCl2, M Na3Cit NaCl, pH 7.0 at +37°C.

The resulting solution volume 16.2 ml has determined the specific activity of FVIII one-step method, equal AndFVIII=304%.

The total number of FVIII in the sample KP, ∑ME=(304%/100)×16.2 ml = 49.2 ME

Output FVIII=49.2/60×1000=817 IU/l AW.

Example 3.

Sample: EWS (volume 2.5 l) with a specific activity equal to 82.5%.

NWS container to grind and put in the bin thermostat. Was unfrozen at +3�B0; C for 4 hours. After the formation of the CP added to the dry sample CaCl2×2H2About equal to 0.92, After 5 min add dry cell trehzameshchenny sodium citrate in the amount of 51.45 g under stirring. After 10 min the mixture of the SPE and KP centrifuged for 15 min at 4500 rpm

The precipitate KP weight of 48.6 g of the centrifuge cups combined pooled, frozen and kept at -20°C.

Output KP, η=48.6 g × 1000/2 .5 l = 19.4 g/l EWS

To determine the specific activity of the obtained KP sample KP weight of 0.54 g was dissolved in a buffer solution of the following composition: M Tris-HCl buffer, pH 7.0, containing M CaCl2at +37°C for 15 minutes to dissolve the KP.

In the resulting solution with a volume of 5.4 ml has determined the specific activity of FVIII one-step method and the concentration of total protein biuret method.

AFVIII=3.79 IU/ml; protein concentration = 8.8 mg/ml

The total number of FVIII in the sample KP, ΣME=(379%/100)×5.4 ml = 20.47 ME

Specific activity of KP Andbeats=3.79/8.8=0.44 IU/mg

The output of FVIII l AW=20.47×19.4/0.54=735 IU/l

The output of FVIII in % of baseline = 735/825=89.1%.

Sources of information

1. Burnouf, T. Chromatography in plasma fractionation: benefits and future trends. J. Chrom. B, 1995, 664, 3-15.

2. Over, J. Plasma Protein Products, Opportunities for the Future. In: Plasma Products Biotechnology Meeting - Extended Reports. Downstream, Special Issue, pp.6-12.

3. Shanbrom, E. Enchance the production of safe plasma preparations. US Pat. No 6541518 (Jan. 2003).

Method enrichment factor VIII cryoprecipitate plasma donor blood, including thawing of fresh frozen plasma blood (NWS) at 1-4°obtaining chriopractic plasma cryosupernatant and cryoprecipitate, characterized in that NWS after the formation of cryoprecipitate add dry calcium chloride in the amount of between 0.001 and 0.004 mol/l, then add dry sodium citrate in an amount of from 0.05 to 0.10 mol/l, stirred for 10-15 min at 1-4°and then the obtained residue cryoprecipitate separated by centrifugation.



 

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