SK8293A3 - Composition for stabilizing bloodplasma during pasteurization and pasteurized plasma solution for therapeutic use - Google Patents

Abstract

The invention relates to a composition intended to stabilise blood plasma during pasteurisation, the blood plasma being whole or devoid of cryoproteins. The composition according to the invention makes it possible to pasteurise very large quantities of plasma (of the order of 60 litres). The invention also relates to the method for pasteurisation of the plasma using the stabilising composition. The pasteurised plasma obtained is intended for therapeutic use.

Description

The invention relates to a composition which makes it possible to stabilize blood plasma during pasteurization, to a method for using the composition and to the plasma solutions thus obtained, which are used in particular for a treatment in which plasma is to be supplemented or blood coagulation factor replaced.

BACKGROUND OF THE INVENTION

In persons who have suffered severe burns or serious injuries or who have undergone major surgery, i. in patients who have experienced a severe fluid loss, treatment involving the addition of full human plasma or plasma that does not contain cryoprotein is used.

This type of treatment typically uses plasma from individual healthy donors who have been pre-screened to avoid the risk of transmission of viral diseases. However, this procedure does not make it possible to eliminate all the risks of viral contamination in the preserological stage, in particular the risk of contamination with various hepatitis viruses and AIDS.

For this reason, it is important to continue to develop methods of virus inactivation and stabilizing agents that maintain the main biological functions of plasma.

It has already been demonstrated that Hepatitis B virus is completely inactivated by heating at 60 ° C for 10 hours in the presence of 0.5 M sodium citrate (Tábor et al., Thrombosis Res. 22,

1981, p. 233-238). However, this procedure leads to a loss of biological activity of some plasma proteins (Tabor et al. And Barrowcliffe et al., Fr. J. Haemotology 55, 1983, pp. 37-46).

It has been shown that it is possible to subject various therapeutically valuable proteins isolated from blood plasma to conventional pasteurization at 60 ° C for 10 hours in the presence of various stabilizing agents. It should be noted, however, that preparations stabilizing one biological activity need absolutely no effect on another biological activity.

These different experimental data suggest that virus inactivation in total plasma (fresh plasma or frozen fresh plasma) or cryoprotein-free plasma cannot be achieved by pasteurization.

Since there is a demand in medicine for total plasma, the present inventors have sought to develop compositions which, during pasteurization, simultaneously guarantee the protection of biological efficacy against denaturation for all therapeutically valuable factors contained in the plasma.

In French patent application no. 90 08375 the inventors have shown that a mixture of sorbitol, heparin, lysine and calcium chloride effectively stabilizes the biological efficacy of plasma factors during the pasteurization process.

However, since this stabilizing formulation, and hence the final formulation, contains heparin, it is not possible to administer this plasma to patients who are already receiving heparin therapy.

For this reason, the inventors have sought to find another preparation which fulfills the same stabilizing function.

SUMMARY OF THE INVENTION

Based on earlier observations that sorbitol gives some degree of protection against thermal denaturation, but the results obtained were different for different plasma samples and the efficacy was not great, the inventors sought to find various additives that, when mixed with sorbitol, would increase its stabilizing ability. Particularly good results were obtained with the addition of sucrose, in an amount of approximately 50%, in relation to the amount of sorbitol.

On the other hand, as observed that calcium chloride is unstable during prolonged heating, it has been replaced by calcium glucanate, which has a good ability to withstand pasteurization conditions.

In addition, the protective effect of the composition is enhanced by the addition of trisodium citrate, provided that its concentration is precisely adjusted because too low a dose allows spontaneous coagulation of plasma, while too high a dose slows down the subsequent activity of coagulation factors during therapeutic use of plasma.

In addition, at least two amino acids, preferably lysine and arginine, are added to the mixture.

Accordingly, the present invention provides a composition for stabilizing blood plasma during pasteurization, characterized in that it is a mixture of sorbitol, sucrose, calcium gluconate, trisodium citrate, lysine and arginine.

For each liter of initial plasma to be pasteurized, the individual components of the formulation are used at the following concentrations:

- sorbitol, 800 to 1400 g, preferably 1300 g,

- sucrose, 400 to 600 g, preferably 514 g,

- calcium gluconate, 3 to 5 mM, preferably 4 mM,

- trisodium citrate, 8 to 25 mM, preferably 15 mM,

- lysine, 1 to 10 g, preferably 5 g,

arginine, 1 to 10 g, preferably 5 g.

The composition of the invention is added to fresh plasma or frozen and thawed fresh plasma, or to plasma that does not contain cryoprecipitate proteins, before the plasma is pasteurized in a liquid state by heating to a temperature of 60 ± 1 ° C for 10 hours.

After pasteurization, the temperature is gradually lowered to 20 ° C and the solution is dialyzed to remove sorbitol and sucrose. The dialysis buffer has a pH of 7.5 and contains 10 mM trisodium citrate, 4 mM calcium gluconate, 0.1 M sodium chloride, lysine at a concentration of 10 g / l and arginine at a concentration of 3 g / l. Dialysis buffers of different compositions may also be used as desired. Thereafter, the solution is concentrated to restore the physiological dose of plasma proteins. The resulting solution is subjected to a clarifying filtration followed by a sterilizing filtration and finally packaged and deep-frozen or lyophilized.

The present invention also provides a plasma pasteurization process, characterized in that the composition according to the invention is added to the plasma prior to heating, and then the pasteurized plasma is dialyzed against the aforementioned buffer.

This method is particularly advantageous since it can be applied to large doses of plasma from several separate donations. These doses may range from 1 liter to several hundred liters. After appropriate control examinations, pasteurization of the batch, if the size is of the order of 60 or more, allows for consistent biochemical quality of the product.

The invention also relates to pasteurized plasma solutions obtained by the process according to the invention, which comprise whole plasma or plasma which does not contain certain proteins (such as cryoprecipitate proteins) and which are intended for therapeutic use, either to replace whole plasma or to compensate for a deficiency. blood factor.

The composition and method of the invention can also be applied to plasma of animal origin. They can advantageously be used, for example, in bovine fetal serum, which is used as an additive for cell cultures, in particular for the manufacture of therapeutic products.

The invention is illustrated in more detail in the following example. This example is illustrative only and does not limit the scope of the invention in any way.

An example of the invention

The following mixture is added to 60 l of molten plasma with gentle stirring:

amount per 1 l of initial plasma sorbitol sucrose calcium gluconate citrate trisodium lysine arginine

1300 g,

514 g, mM, mM, g,

g.

The pasteurization is carried out in a thermostatic tank for 10 hours at ° C. After pasteurization, the temperature is gradually lowered to 20 ° C and then the solution is dialyzed to remove sorbitol and sucrose. The dialysis is carried out, for example, in a cassette ultrafiltration system (Pellicon ( ^R )). The dialysis buffer used has the following composition:

trisodium citrate 10mM lysine 10g / 1 arginine 3g / 1 calcium gluconate 4mM sodium chloride0,1 M

Sorbitol removal is checked by colorimetric analysis and sucrose removal is checked by enzymatic analysis of the resulting product.

Dialysis may be followed, if necessary, by a concentration procedure carried out in the same apparatus. In this procedure, the coagulation factor content is again set to a value of approximately 1 U / ml, which is a value of quality plasma for therapeutic use. The product is dialyzed at an osmotic pressure of 370 mosmol / l and at a pH of 7 to 7.5.

The product is sterilized by filtration, e.g., using a filter material ^Rf ^ Millipack 40 (Millipore ^(R)) with a pore size of 0.22 μιη. The obtained product is packaged in plastic bags or bottles and frozen or lyophilized.

The effectiveness of pasteurization in virus inactivation is evaluated using a variety of viruses, the use of which is recommended by a group of experts in biotechnology and pharmacology of the European Community authorities for evaluating procedures aimed at eliminating and inactivating viruses in biological products. The results are shown in the following table.

virus gene structure decrease duration of infectivity (log 10)

HIV RNA E > 5 <10 min Polio Sabin 2 RNA NE > 6.23 <5 h Vaccine DNA E > 4.30 <1 h Herpes- DNA E > 4.15 > 5 h aujeszky parainfluenza RNA E > 6.30 <2 h type 3 Reovirus type 3 RNA NE > 3.28 <10 h Sindbis RNA E > 5.74 <5 h

Notes: E = with cover

NO = without packaging

The quality control results for six consecutive pilot batches are shown in the following tables.

Table II

Regeneration of blood factors

concentration (U / ml) Contents (%) proteins (g / l) 60.0 ± 106 93 ± 2 Factor VIII: c 1.10 ± 0.26 94 ± 7 (IU / ml) Factor VIII: c 0.70 ± 0.09 83 ± 13 (Chromogenic) Factor V: c 1.12 ± 0.25 83 ± 4 Factor XI: c 0.98 ± 0.06 88 ± 7 Factor IX: c 0.64 ± 0.10 59 ± 7 Factor VII: c 0.65 ± 0.05 67 ± 6 fibrinogen Ag (g / l) 3.10 ± 0.28 92 ± 5 clotting fibrinogen (g / L) 2.50 ± 0.17 92 ± 3 antithrombin activity III (g / L) 0.99 ± 0.04 92 ± 3 α-1-antitrypsin activity (g / l) 0.96 + 0.10 92 + 3 APTT 35-38 seconds

Table III Quality control: absence of proteases and degradation products concentration (U / l)

Thrombin <0.01

PKA (%) <2 kallikrein (%) 3 factor IXa (U / ml) <0.1 »

Table ΙΙΙ-continued factor Xa (U / ml) <0.1 factor VIIb / FVIIa0.97 proteolytic activity 0.03 (P.2288 - OD / mn) fibrinogen degradation products (ng / ml) <250

Table IV

In vivo quality control in animals Test in rats:

blood pressure heart rate toxicity (intravenous) injection - 25 ml / kg) rabbit test:

pyrogenicity <-2% without change is not toxic pyrogen-free

Plasma pasteurized in the presence of the above stabilizing agent complies with the standards that are imposed on plasma for therapeutic use.

- 10 blood with t

PV & - <33

Υ plasma during v in m. that is creature-

Claims (17)

A pasteurization stabilizing composition, characterized by a mixture of sorbitol, sucrose, calcium gluconate, trisodium citrate, lysine and arginine. 2. A formulation according to a concentration ranging from 800 to 1400 1, characterized in that sorbitol is present in the initial plasma g / l. i s lies Composition according to claim 2, characterized in that the concentration of sorbitol is 1300 g / l. Composition according to any one of claims 1 to 3, characterized in that the concentration of sucrose is in the range of 400 to 600 g / l. The composition of claim 4, wherein the sucrose concentration is 514 g / L. A composition according to any one of claims 1 to 5 wherein the concentration of calcium gluconate is in the range of 3 to 5 mM. The composition of claim 6, wherein the calcium gluconate concentration is 4 mM. Composition according to any one of claims 1 to 7, characterized in that the trisodium citrate concentration ranges from 8 to 25 mM. The composition of claim 8, wherein the trisodium citrate concentration is 15 mM. Composition according to any one of claims 1 to 9, characterized in that the concentrations of lysine and arginine are in the range of 1 to 10 g / l. Composition according to claim 10, characterized in that the concentrations of lysine and arginine are 5 g / l. 12. A process for the pasteurization of blood plasma comprising adding to the plasma prior to the heating step a composition according to any one of claims 1 to 11 and after the heating step removing sugars from the mixture by dialysis against a buffer containing trisodium citrate, calcium gluconate, sodium chloride. , lysine and arginine. Method according to claim 12, characterized in that a dialysis buffer containing 10 mM trisodium citrate, 4 mM calcium gluconate, 0.1 M sodium chloride, 10 g / l lysine and 3 g / l arginine is used. 14. A method according to claim 12 or 13, which is applicable to a plasma volume in the range of 1 liter to several hundred liters. Complete plasma for therapeutic use, pasteurized by a process according to any one of claims 12 to 14. Cryoprecipitated plasma supernatant for therapeutic use, pasteurized by a process according to any one of claims 12 to 14. Plasma or serum of animal origin pasteurized by a process according to any one of claims 12 to 14.