METHOD FOR PREPARING A CONCENTRATED SOLUTION OF FACTOR VIII.

Abstract

L'invention concerne un procédé de préparation d'un produit tel qu'obtenu par le procédé selon la revendication 1 du brevet principal, qui comprend les étapes suivantes: a) on solubilise un cryoprécipité; b) on soumet la solution obtenue à une première précipitation en ajustant le pH à une valeur comprise entre 6,7 et 7,3 en ajoutant un polysaccharide sulfate (SPS) dans des conditions de température et de concentration aptes à prévenir la précipitation du facteur VIII, et permettant essentiellement la précipitation du fibrinogène et de la fibronectine tout en laissant le facteur VIII en solution; c) on sépare le culot du surnageant; d) on soumet le surnageant à une seconde précipitation en ajoutant un gel d'hydroxyde d'aluminium en quantité comprise entre 100 et 200 g / kg de cryoprécipité de départ, en ajustant le pH à une valeur comprise entre 5.8 et 6.3, de façon à précipiter essentiellement les facteurs vitamine K dépendants et les autres contaminants protéiques tout en laissant le facteur VIII en solution; e) on sépare le culot du surnageant; f) on récupère le surnageant qui est une solution concentrée de Facteur VIII.

Description

I. The present invention relates to a method for preparing a solution concentrated factor VIII of intermediate purity from a cryoprecipitate and aims to constitute a certificate of addition on demand French patent filed on March 15, 1990 under No. 90 03328. The process described in the main patent application comprises the following steps: a) a cryoprecipitate is solubilized; b) the resulting solution is subjected to a first precipitation by adding a sulfated polysaccharide (SPS) in an amount of 5 to 20 U / ml of solution, adjusting the pH to a value between 6.7 and 7.3, and lowering the temperature to a value between 10 and 16 Wc; c) the pellet is separated from the supernatant; d) the supernatant is subjected to a second precipitation by adding an aluminum hydroxide gel in an amount of 100 to 200 g / kg of the initial cryoprecipitate, adjusting the pH to a value between 5.8 and 6.3; e) the pellet is separated from the supernatant; f) the supernatant, which is a concentrated solution, is recovered of Factor VIII. It has now been observed that it is possible to obtain a concentrated solution of factor VIII, in the absence of factor VIII precipitation, under the operating conditions of temperature and quantity of SPS. during step b) which vary in a significantly wider range. Therefore, the purpose of this certificate of addition is a process for preparing a product as obtained by the process according to claim 1 of the main patent, which includes the following steps: a) a cryoprecipitate is solubilized, b) the resulting solution is subjected to a first precipitation by adjusting the pH to a value between 6.7 and 7.3, by adding a sulfated polysaccharide (SPS) under temperature and concentration conditions suitable for preventing the precipitation of factor VIII, and essentially allowing the precipitation of fibrinogen and fibronectin while leaving factor VIII in solution, c) the pellet is separated from the supernatant;d) the supernatant is subjected to a second precipitation by adding an aluminium hydroxide gel in an amount between 100 and g / kg of starting cryoprecipitate, adjusting the pH to a value between 58 and 63, so as to essentially precipitate the vitamin K dependent factors and other protein contaminants while leaving factor VIII in solution; e) the pellet is separated from the supernatant; f) the supernatant, which is a concentrated solution, is recovered; of Factor VIII. Thus, by appropriately choosing the parameters of step b), it is possible to work at higher temperatures, with quantities of SPS greater than those envisaged in the application. main, without reducing the yield in factor VIII. According to an advantageous embodiment of the invention, in step b), the SPS is added in an amount between 5 and 40 U / ml of solution and the temperature is between 5 and 251°C. Preferably, the SPS is added in an amount equal to 24 U / ml and the temperature is [temperature missing]. Among the usable sulfated polysaccharides (SPS), examples include polysulfated mucopolysaccharide, pentosan polysulfate, chondroitin sulfate, dextran sulfate, and heparin. will preferably use heparin. If applicable, the process also includes a step final stage of viral inactivation. According to a further feature of the invention, the process is characterized by obtaining a factor VIII whose activity specific is less than 1.5 IU / mg. The following example illustrates the invention. 1) Preparation of the cryoprecipitate: Human blood collected in the presence of sodium citrate is centrifuged to remove the cellular fraction. The resulting plasma is frozen at -40°C in plastic bags. The cryoprecipitate is then obtained in three successive steps: Pre-thawing of the plasma: it is gradually warmed- ment so that its temperature drops from -40°C to -1°C in 8 hours; thawing of the plasma: the plastic bags are removed, then the frozen plasma is placed in a grinder for approximately 2 minutes. The temperature of the resulting solution is maintained between 0 and 0.5°C; separation of the cryoprecipitate: the suspension is centrifuged at a temperature between 1 and 4 C. The cryoprecipitate is analyzed below. The values ​​are reported relative to plasma, i.e., 6.5 g of cryoprecipitate. Before heparin precipitation: Factor VIII(c) (U) 346 5 Total protein (mg) 840 Specific activity of Factor VIII (U / mg) 0 41 Fibrinogen (mg) 410 Fibronectin (mg) 50 2) Preparation of a concentrated Factor VIII solution: g of cryoprecipitate are resuspended in four times its weight volume of 20 mM Tris-HCl buffer at pH 7. The pH is then adjusted to 7.1 with 0.5 N acetic acid. Standard heparin, commercially available from LEO France, is then added at a concentration of 24 U / ml of solution, and the temperature is set at 20°C. To recover the supernatant, centrifugation is performed at 4000 t / min for 15 minutes. A quantity of Al(OH)3 corresponding to g / kg of starting cryoprecipitate is added to the supernatant, and the pH is adjusted to 6.0 with 0.5 N acetic acid. As before, centrifugation is carried out at 4000 rpm for 15 minutes to recover the supernatant. 3) Viral inactivation of the Factor VIII concentrated solution: 1 mM CaCl2 is added to the resulting concentrated solution, and the pH is adjusted to 7 with 0.1 N NaOH. Next, a viral inactivation mixture is added to the solution to achieve a final concentration of 1% Tween 80 and 0.3% TnBP (volume / volume). lume) The solution is incubated for 6 hours at 24 C. 4) Test Methods a) Determination of Factor VIII content: by chromogenic assay after neutralization of excess heparin; b) Determination of total protein content using reagents marketed by Bio-Rad; c) Determination of fibrinogen content using Behring reagents (simple radial immunodiffusion); d) Determination of fibronectin content: using Behring reagents (simple radial immunodiffusion); e) Determination of heparin content: by chromogenic assay. The following results are obtained, as reported in the table below: Initial, After, After, After, Precipitation, After the Second, After Inactivation. Parameter: Solubilization due to heparin, Viral precipitation, Cryoprecipitate. Yield (in 100): 91, 85, 100, Factor VIII (96). Concentration in total protein 23 13.5 6.7 - (mg / ml) Elimination rate 41.4 50.4 - Proteins (%) Specific activity of factor VIII 0.41 0.61 1.05 1.05 (U / rmng) Ui cn w IX U'

Claims

DEMANDS 1. A process for preparing a product as obtained by the process according to claim 1 of the main patent, comprising the following steps: a) a cryoprecipitate is solubilized; b) the resulting solution is subjected to a first precipitation by adjusting the pH to a value between 6.7 and 7.3 by adding a polysaccharide sulfate (SPS) under temperature and concentration conditions suitable for preventing the precipitation of factor VIII, and essentially allowing the precipitation of fibrinogen and fibronectin while leaving factor VIII in solution; c) the pellet is separated from the supernatant; d) the supernatant is subjected to a second precipitation by adding an aluminum hydroxide gel in an amount between 100 and 200 g / kg of starting cryoprecipitate, adjusting the pH to a value between 5.8 and 6.3, so as to essentially precipitate the vitamin K-dependent factors and other protein contaminants while leaving factor VIII in solution;e) the pellet is separated from the supernatant; f) the supernatant, which is a concentrated solution, is recovered; of Factor VIII.

2. A process according to claim 1, characterized in that in step b), the SPS is added in an amount between 5 and 40 U / ml of solution and the temperature is between 5 and 250°C.

3. A process according to claim 2, characterized in that the SPS is added in a quantity equal to 24 U / ml and the temperature is 20°C.

4. A method according to any one of claims 1 to 3, characterized in that the factor VIII obtained has a specific activity of less than 1.5 IU / mg.