LT3226B - Composition for stabilizing blood plasma during pasteurisation 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 encompasses a composition for stabilizing blood plasma during pasteurisation, a process for pasteurization using said composition, and plasma solutions obtained therefrom, in particular, for plasma transfusion and for coagulation factor therapy.

All human plasma, or plasma without cryoprotein, is still used in transfusion therapy for persons suffering from severe burns or serious injuries, or who have undergone major surgical interventions, generally in cases of high fluid loss.

Commonly used for this type of treatment is plasma obtained from individual healthy individuals previously screened to prevent any risk of transmission of viral diseases. However, this procedure does not ensure complete elimination of the risk of viral contamination in the pre-serological stage, especially with various hepatitis and AIDS viruses.

Therefore, it is important to continue the search for viral inactivation methods and stabilization compositions that protect critical biological functions of plasma.

Hepatitis B virus has been shown to be completely inactivated for 10 hours. 0.5 M sodium citrate at 60 ° C (Tabor et al., Thrombosis Res. 22, 1981, 233-238). However, this treatment results in the loss of biological activity of some plasma proteins (Tabor et al. And Barrowcliffe et al., Fr. J. Haemotology 55, 37-46, 1983).

It has been demonstrated that various proteins having therapeutic value can be pasteurized from plasma

Usually 10 hours. At 60 ° C in the presence of various stabilizing agents. However, it is noted that compositions that stabilize one species's biological activity may have no effect on another species's biological activity.

These different conclusions from the experimental data give the impression that viral inactivation of whole plasma (fresh plasma or frozen fresh plasma) or plasma without cryoprotein cannot be achieved by pasteurization.

Nevertheless, since there is a medical need for whole plasma, the Applicant has sought compositions that provide simultaneous protection of the biological activity of all plasma factors of therapeutic value from denaturation during pasteurization.

Thus, French patent application no. Applicant 90 08 375 demonstrated that a mixture of sorbitol, heparin, lysine and CaCl ₂ effectively stabilized the biological activity of plasma factors during pasteurisation.

However, due to the presence of heparin in this stabilizing mixture and in the same end product, patients already treated with heparin should not be injected with this plasma.

That is why the applicant was looking for another composition that performs the same stabilizing function.

Having previously noticed that sorbitol to some extent enhances protection against thermal denaturation, but results vary between plasma samples and efficiency is not high, the applicant sought various additives that, when mixed with sorbitol, would increase the stabilizing power of the mixture; especially good results were obtained when sucrose was added to the mixture in an amount corresponding to approximately 50% of the sorbitol content.

On the other hand, when it was found that CaCl _{2 was} not stable after prolonged heating, it was replaced by calcium gluconate, which exhibits good resistance under pasteurisation conditions.

In addition, the addition of trisodium citrate enhanced the protective effect of the mixture if its concentration was accurately controlled: too low a dose results in spontaneous plasma coagulation, whereas an overdose reduces subsequent coagulation factor activity during therapeutic use of plasma.

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

Thus, the stabilizing composition of the present invention consists of a mixture of sorbitol, sucrose, calcium gluconate, trisodium citrate, lysine and arginine. The following concentrations (1 liter of starting plasma) are used for different plasma pasteurization additives:

- Sorbitol, 800-1,400 g, preferably 1,300 g

- sucrose, 400-600 g, preferably 514 g

calcium gluconate, 3-5 mM, preferably 4 mM

trisodium citrate, 8-25 mM, preferably 15 mM

- lysine, 1-10 g, preferably 5 g

- arginine, 1-10 g, preferably 5 g.

The composition of the present invention is added to fresh plasma or to frozen and thawed fresh plasma or to plasma containing no cryoprecipitate proteins, e.g.

prior to pasteurization of said plasma by heating in liquid state for 10 hours. At 60 ° C ± 1 ° C.

After pasteurization, the temperature is gradually lowered to 20 ° C and the solution is dialyzed to remove sorbitol and sucrose. The pH of the dialysis solution is 7.5 and consists of 10 mM trisodium citrate, 4 mM calcium gluconate, 0.1 M sodium chloride, 10 g / l lysine and 3 g / l l arginine. Dialysis buffers of various compositions may also be used on request. The solution is then concentrated to restore the physiological normalization of plasma proteins. The resulting solution is filtered and then distributed and stored frozen or lyophilized.

The invention also relates to a method of plasma pasteurisation which comprises placing the composition of the present invention in plasma prior to heating, followed by dialysis of the pasteurized plasma against the buffer described above.

This method is particularly useful because it can be applied to large batches of plasma from several individual sources and can range from 1 liter to several hundred liters. Batches of 60 liters or more may be subjected to pasteurisation after appropriate controls, thereby guaranteeing the appropriate biochemical quality of the product.

The invention also encompasses pasteurized plasma solutions obtained by the invention, said solutions including all plasma or plasma without some proteins (such as cryoprecipitate proteins) for therapeutic use as well as for replacement of whole plasma or to compensate for single blood factor deficiency.

The composition and method of the present invention are thus adapted to animal plasma. They may be usefully adapted, for example, to the fetal pat, for example, as serum for use as an adjunct to cell cultures, mainly for the preparation of therapeutic products.

The following example illustrates an embodiment of the present invention without limiting its scope.

Example:

The following mixture is added to 60 liters of thawed plasma with gentle mixing:

volume per liter of plasma sorbitol 1,300 g sucrose 514 g calcium gluconate 4 mM trisodium citrate 15 mM lysine 5 g arginine 5 g

The pasteurisation is carried out in a thermostated dish for 10 hours. At 60 ° C.

After pasteurization, the temperature is gradually lowered to 20 ° C and then the solution is dialyzed to remove sorbitol and sucrose.

Dialysis is performed using, for example, a cassette ultrafiltration system (Pellicon®).

The composition of the dialysis buffer is as follows:

- trisodium citrate 10 mM

- lysine 10 g / ltr

- arginine 3 g / ltr

- calcium gluconate 4 mM

- Sodium chloride 0.1 M

Sorbitol removal is controlled colorimetrically and sucrose is controlled by enzymatic analysis of the final product.

Dialysis may be followed, if necessary, by concentration using the same Equipment to restore coagulation factor levels to approximately 1 U / ml as in good quality therapeutic plasma. The product is dialyzable at 370 mosmol / l osmotic pressure and pH 7-7.5.

The product is then sterilized by filtration, eg using Millipack® 40, Millipore® with 0.22 μ pores. The product is then put into plastic bags or bottles and frozen or lyophilized.

The efficacy of viral inactivation during pasteurisation was evaluated by selecting viruses recommended for ratification by the European Panel of Biotechnology / Pharmacy Specialists for the elimination and inactivation of viruses in biological products. The results are shown in Table I.

Table I

The virus Gene Structure- volume Infectious bite decrease (log 10) Duration HIV RNA E > 5 <10 min POLIO SABIN 1 RNA NO > 6.23 <5 or. VACCINE DNA E > 4.30 <1 or. HERPES-AUJESZKY DNA E > 4.13 <5 or. PARA Flu Type 3 RNA E > 6.3; <2 or. REOVIRUS Type 3 RNA NO > 3.28 <10 or. SINDBIS RNA E > 5.74 <5 or.

E - with cover NO - without cover

The quality control results obtained in 6 consecutive controlled batches are shown in the following tables.

Table II.

Blood Factor Recovery

KDNCENTPATION, U / ml OUTPUT% Proteins (g / ltr) 60.0 + 10.6 93 + 2 Factor VIII: C (IU / ml 1.10 + 0.26 94 + 7 Factor VIII: C (chromogenic) 0.70 + 0.09 83 + 13 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 / ltr) 3.10 + 0.28 92 + 5 Coagulating fibrinogen (g / ltr) 2.50 + 0.17 92 + 3 Antithrombin III activity (g / ltr) 0.99 + 0.04 92 + 3 For alpha-1-antitrypsin active substances (g / ltr) 0.96 + 0.10 92 + 3 APTT 35-38 seconds -

Table III.

Quality control: absence of protease and degradation products

KDNCENTRATION (U / L) Thrombin <0.01 PKA (%) <2 Calicrine (%) 3 Factor IXa (U / ml) <0.1 Factor Xa (U / ml) <0.1 Factor VIIb / F Villa 0.97 Proteolytic activity (S. 2288 - OD / mn) 0.03 Fibrinogen degradation products (ng / ml) <250

Table IV.

Quality control in vivo

Rat test:

- Blood pressure

- Pulse

Toxicity (intravenous injection: 25 ml / kg

Test with rabbits:

- Pyrogenicity <-2% unchanged toxicity is not pyrogenic

Plasma pasteurized with the stabilizing composition described above meets the standards established for therapeutic use.

Claims (12)

DEFINITION OF INVENTION A composition for stabilizing blood plasma during pasteurisation, characterized in that it consists of a mixture of sorbitol, sucrose, calcium gluconate, trisodium citrate, lysine and arginine. Composition according to claim 1, characterized in that the concentration of sorbitol is 800-1400 g / l of primary plasma, preferably 1300 g / l. Composition according to claim 1 or 2, characterized in that the sucrose concentration is 400600 g / l and preferably 514 g / l. 4. The composition according to any concentration is 3-5 mM, 5. The composition according to any concentration is 8-25 mM, The composition according to any one of claims 1-3, wherein the concentration of calcium gluconate is preferably 4 mM. A process according to any one of claims 1-4, wherein the trisodium citrate is preferably 15 mM. Claims 1-5 wherein lysine and arginine, preferably 5 g / l. 7. A process for the pasteurization of blood plasma comprising the step of introducing a composition according to any one of claims 1 to 6 into the plasma prior to the heating step and the sugar being removed from the composition by dialysis against the buffer containing trisodium citrate, calcium gluconate, sodium chloride, lysine and arginine. 8. The bud of claim 7, wherein the dialysis buffer contains 10 mM trisodium citrate, 4 mM calcium gluconate, 0.1 M sodium chloride, 10 g / l lysine and 3 g / l arginine. 9. A method according to claim 7 or claim 8, characterized in that it is applied to plasma volumes of from 1 to several hundred liters. 10. All plasma for therapeutic use, other than being pasteurized, 10 using the method of any of claims 7-9. 11. Cold precipitated plasma supernatant for therapeutic use, characterized in that it has been pasteurized using Method 15 according to any one of claims 7-9. Plasma or animal serum characterized in that it has been pasteurized using the method of any of claims 7-9.