RU2000113224A - METHOD FOR PRODUCING A HIGH DEGREE OF VIRUS-SAFE COMPONENTS FOR PRODUCING FIBRINE GLUE FROM A HUMAN PLASMA POOL - Google Patents

Claims (16)

1. The method of obtaining thrombin, comprising the following stages
a) precipitation of whole plasma proteins by adding the first salt in an amount sufficient to achieve the salting-out effect and to bring the pH to a value of from about 7.5 to about 8.5 at a temperature of from about 0 ^o C to about 4 ^o C, or by adding a second salt in an amount sufficient to achieve acid precipitation and to bring the pH to a value of from about 4.5 to about 5.5 at a temperature of about 4 ^{° C.} to about 20 ^{° C.} , whereby fibrinogen precipitates, a factor XIII and fibronectin, moreover, the specified precipitation pr bred in the presence of amino-6-hexanoic acid in a concentration of at least 50 mm; and isolating a prothrombin-containing supernatant;
b) diafiltration of the indicated supernatant until a prothrombin solution is obtained that has an osmolality of about 100 mOsmol / kg or less;
c) diluting said prothrombin solution with water, which is added in relation to about 4 volumes of water per 1 volume of prothrombin solution;
d) prothrombin precipitation by adding an acid solution until a pH of about 5.2 is obtained;
e) solubilizing the precipitate of step (d) in a solution having about a neutral pH and
e) the conversion of prothrombin stage (d) to thrombin in the presence of calcium chloride to achieve a concentration of calcium chloride from about 20 to about 32 mm. 2. The method according to p. 1, which, in addition, includes the following stages
g) incubating said thrombin with a viricidal solution of solvent / detergent in an amount sufficient to inactivate lipid-containing viruses;
h) purification of incubated trobin by subsequent ion-exchange chromatography using one cation-exchange medium with a sulfalkyl-activated polysaccharide selected from the group consisting of sulfagyl-activated polyagarose, sulfide-activated polydextran and an incompressible composition medium consisting of sulfonyl-activated dextran and particles of silicon dioxide, having a high degree of selectivity with respect to thrombin, and using an aqueous salt solution as an eluting agent of a solution of at least three and increasing concentrations
i) isolating the thrombin peak eluate during chromatography (h) and replacing the eluate salt with a physiologically acceptable stabilizing buffer of the preparation in order to stabilize the thrombin isolated, and preparing the thrombin buffered solution.  3. The method according to p. 1 or 2, where the step of diafiltration (b) includes replacing water in a volume equivalent to four volumes of supernatant.  4. The method according to p. 1 or 3, where in stage (e), 40 mM calcium chloride is added as a solution in a ratio of about 4 volumes per 1 volume of solubilized precipitate of stage (d).  5. The method according to p. 2, where in stage (e) as a solution add 40 mm calcium chloride in relation to about 4 volumes of calcium chloride per 1 volume of solubilized precipitate of stage (d).  6. The method according to p. 2, 3 or 5, where the cation exchange medium is basically an incompressible composite medium containing activated sulfoalkyl dextran and particles of silicon dioxide.  7. The method of claim 6, wherein the cation exchange medium is a substantially incompressible composite medium comprising sulfopropyl activated dextran and silica particles.  8. The method according to any one of paragraphs. 2, 3 and 5 - 7, in addition, comprising stage (k), comprising filtering the buffer solution of the thrombin preparation through a membrane consisting of hollow fibers of copper-ammonia cellulose, in order to filter out the virions present in the buffer solution of the drug, and obtaining mostly free virion thrombin buffer solution.  9. The method of claim 8, wherein said hollow fiber membrane of copper ammonia cellulose has a pore size of preferably about 15 nm.  10. The method according to p. 8 or 9, which, in addition, includes the following stages l) lyophilization of the thrombin solution obtained in stage (k), and m) dry heating the lyophilized thrombin preparation to inactivate any remaining virions without thrombin denaturation.  11. The method according to PP. 2, 3, 5, 6 or 7, which, in addition, includes the final stage of lyophilization to dryness of the thrombin preparation buffer solution and heat treatment of this solution to obtain undenatured thrombin essentially free of virions. 12. The method according to p. 10 or 11, where the specified stage of dry heat treatment is carried out, preferably by heating the lyophilized product at about 100 ^o C for about 1 to 2 hours  13. The method according to any one of paragraphs. 2, 3 and 5-12, where the buffer solution of the drug includes an aqueous solution of citrate, sodium chloride, Tris-HC1 and serum albumin at a pH of about 7.3 in quantities sufficient to stabilize thrombin in order to prevent a significant loss of its activity in the process heat treatment.  14. The method according to p. 13, where the buffer solution of the drug includes an aqueous solution containing about 0.25% sodium citrate, 0.45% sodium chloride, 0.25% Tris-HCl (all values are given in wt./about %) and serum albumin in an amount that is approximately 20 times higher than the total amount of protein in the eluate of the thrombin peak and which is adjusted to 2% wt. before lyophilization. /about. ; and having a pH of about 7.3.  15. The method according to any one of paragraphs. 2, 3 and 5-14, wherein said at least three increasing salt concentrations in aqueous saline are substantially about 0.08 M, 0.15 M and 0.4 M NaCl in human thrombin buffer.  16. The method according to any one of paragraphs. 2, 3 and 5-15, where the incubation step (g) is carried out in the presence of a viricidal solvent / detergent solution that does not contain sodium citrate.