SK278348B6 - Preparation method of blood albumin - Google Patents

Abstract

A protein solution of the fraction IV by Cohn,in pyrogenous water with protein concentration of 10 up to 100 grams per one litre, is precipitated, at the pH value of 5,8 ± 0,3 with the use of ethanol to the total ethanol concentration of 40 ± 10 percent of the total volume closed to the reaction mixture, at the temperature of -5 Celsius degrees ± 0,5 Celsius degrees, while the vast proteins having no albumin character are coagulated as the suspension being separated with the use of whizzer, the supernatant is filtered and albumin is precipitated of that, at ethanol concentration of 40 ± 10 percent of the total volume closed to the reaction mixture, at the pH value of 4,8 ± 0,3, at the temperature of -14 ± 4 Celsius degrees, and after that the albumin paste is separated as the fraction V by Cohn, with the use of a whizzer, it is brought into the solution and the low molecular compounds are removed with the use of the diafiltration or ultrafiltration.

Description

Technical field

The invention relates to a process for the preparation of blood albumin. Current technological practice of ethanol plasma or serum fractionation is oriented towards the production of albumin predominantly from fraction V according to Cohn et al. This technological process for producing albumin results in an albumin-containing waste fraction, such as Cohn et al. Waste fraction IV, wherein the protein portion of waste fraction IV, isolated while maintaining the technological fractionation parameters, contains approximately 10 to 25% w / w% albumin. If the technological parameters are not observed, the content of albumin can reach 30 to 75 m / m%. It is convenient and economical to utilize this scrap albumin content for the preparation of a therapeutic albumin preparation.

BACKGROUND OF THE INVENTION

Until now, the preparation of blood albumin from liquid plasma or serum is predominantly with ethanol / Cohn EJ, Strong LE, Huges WL, Mulford DJ, Ashworth JN, Melin M., Taylor ML: J.Amer. Chem. Soc. 68,459,1946; Taylor HL, Bloom FC, Mc Call KB, and Hyndman LA: J. Amer. Chem. Soc. 78,1353,1956; Krijnen HW, Mastenbroek GGA: Vox Sang. 2,405,1957; Mulford, D., Mealay, EH, Welton, LD: J. Clin. Invest. 34,983, 1955; Nitschmann HS, Kistler P., Lergier W., Helv. Chim. Acta 37,866,1954; Nitschmann, HS, Kistler, P., Reufer, H., Hässig, A., Joss, A .: Vox Sang. 1183.1956; Kistler, P., Nitschmann, HS: Vox Sang. The use of ethanol together with Zn ⁺² and Ba ⁺² has been described (Cohn EJ, Gurd FNR, Surgenor DM, Bames BA, Brown RK, Derounaux G., Gillespie JM, Kahnt FW, Lewer WF, Liu CH, Mittelman D., Mouton RF, Schmid K., Uroma E., J. Amer. Chem. Soc. 72,465,1950 (as well as the use of Zn ⁺²⁾ (Cohn EJ, Tullis JL, Surgenor DM, D'Hont M .: Science 114,479,1951).

Efforts to increase albumin recovery have led albumin manufacturers to introduce heat-denaturing methods (Hoch H., Chanutin, A .: Árch. Biochem. Biophys. 51,27,1954 ,; Lundblad, J.I .: Vox Sang. 5,122,1960; Schneider W., Lefevre H., Mc Carty L. J., Fiedler H., Blut 30,121,1975].

Efforts have been made to avoid the technological practice of using denaturing methods around 70 ° C. It has been found that the addition of chloroform reduces the denaturation temperature of non-albumin impurities! up to 40 ° C to 50 ° C / Libich M .: Cs. U.S. Patent 129936; Libich M .: AO no. 210088; Libich M., Novotny J., Ulrych S .; AO č. 239715 /. Chloroform addition was also used to precipitate lipoproteins from human albumin solution / Pokorny M., Foukal Z., Pardon J .: AO no. 194957 /.

Recent knowledge of the effects of chloroform does not recommend its use. Chloroform is also present in drinking water, at about 1 mg per liter, where chlorination by the action of methane dissolved in water produces trace amounts and is considered to be a mutagenic compound / Chen AM, Skochdopole J., Koski P., Cóle L .: Science 207,90,1980]. Although the use of chloroform in anesthesia has been common in the recent past, this is currently not recommended (Jones I.W .: Anaesthesia 3 8,578,1983).

The effect of chloroform on human blood plasma proteins is very specific and affects the properties of these proteins, as well as the immune system of the human and animal organisms in a wide range, with the most hepatotoxic and carcinogenic effects 5 / Štcfanovič J., Elder Z.,

J. Hyg. Epidemiol.Microbiol. Immunol. 31,1,1987; Štefanovič J., Murgašová I., Absolonová O., Žalkovičová M .: Bratislav.l.listy 89, No.3, 160.1988 /. The use of chloroform for albumin isolation while partially remaining 10 is the recovery of albumin, but results in the binding of part of chloroform to albumin in an amount of up to 500 ± 50 mg of chloroform per 1000 ml of 20m / m% albumin which cannot be removed with gel filtration or diafiltration; ultrafiltration.

The disadvantages of the production processes used so far in recovering albumin from waste fraction IV are eliminated by the production process according to the present invention, which can be used to prepare albumin with an electrophoretic purity of 96.5 ± 0.5% and a yield of about 2.0 ± 0.5 g per liter of starting plasma or serum. If the technological parameters of insulation of fraction IV according to Cohn et al. the recovery of albumin may be substantially higher.

The production process does not require the addition of chloroform 25 mu or denaturing temperatures above 0 ° C, which makes it possible to further process the waste proteins if necessary. It is characterized by a reduction in recovery yields of albumin that would be different than co-precipitated in the waste fraction IV according to Cohn et al. The therapeutic preparation contains chloroform, which has been analytically confirmed.

SUMMARY OF THE INVENTION

The essence of the process for the preparation of blood albumin consists in bringing a protein paste, for example Fraction IV, into solution. First, nonalbumin impurities are eluted with ethanol, which are removed, for example, by centrifugation, and then ethanol is precipitated with albumin as fraction V, which, after separation, for example by centrifugation, is brought into an aqueous solution, clarified by filtration, freed from low molecular compounds by diafiltration or ultrafiltration; undergo further processing into a therapeutic preparation.

The invention is further elucidated by way of example of an embodiment of the invention, the scope of which is neither limited nor exhaustive.

DETAILED DESCRIPTION OF THE INVENTION

For example, the starting material may be a fraction IV paste, whether or not the technological fractionation parameters are observed according to the method of Cohn et al. This albumin-containing proteinaceous material is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to 6 ° C or in distilled pyrogen-free water where 60 has been adjusted to an ionic strength of 0.1, for example by adding sodium chloride to concentrate the proteins. in the range of 10 to 100 g per liter. Dissolution is accelerated by stirring and adjusting the pH to 7.4 + 0.1 at 0 ° C to 6 ° C. In technological conditions, the above conditions are generally maintained when 1 kg of the fraction is dissolved

SK 278348 Β6

IV in 5000 ml of pyrogen-free distilled water or in distilled pyrogen-free water with an ionic strength of 0.1, for example from sodium chloride, at 0 ° C to 6 ° C.

The precipitation of ballast nonalbumin proteins is carried out with 40 + 10% ethanol from the total volume of the reaction mixture at a final temperature of -5 ° C + 0.5 ° C, at a pH of 5.8 + 0.3. The ballast non-albumin proteins are precipitated in the form of a fine suspension which is stirred at the indicated temperature for, for example, 3 to 12 hours, then separated, for example, by centrifugation, and the supernatant obtained is subjected to a black filtration with diatomaceous earth.

The albumin is precipitated from the supernatant after filtration with ethanol to a total concentration of 40 + 10% of the total volume of the reaction mixture at pH 4.8 + 0.3 at -14 ° C + 4 ° C whereupon the albumin precipitates as a fine suspension which The mixture is stirred at said temperature for, for example, 3 to 12 hours, then separated, for example, by centrifugation, and the albumin protein paste is obtained in fraction V, which is subjected to further processing.

Albumin paste protein material as a fraction

V is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to 6 ° C such that the protein concentration is between 10 and 150 g per liter at a pH between 4.0 and 8.0, subjected to a black filtration, % of the low-molecular weight substances by diafiltration or ultradiafiltration, and the albumin solution thus obtained is subjected to further processing. In technological conditions, the above conditions are generally maintained by dissolving 1 kg of albumin paste in 2000 ml of distilled pyrogen-free water at a temperature of 0 ° C to 6 ° C.

Qualitative and quantitative parameters of blood albumin for therapeutic purposes are determined by the applicable company standard or by valid pharmacopoeia.

Claims (4)

PATENT CLAIMS Process for the preparation of blood albumin, characterized in that the waste fraction IV according to Cohn is dissolved in such an amount of pyrogen-free water at a temperature of 0 ° C to 6 ° C or in pyrogen-free water where the ionic strength has been adjusted to 0.1, preferably by adding sodium chloride so that the protein concentration is in the range of 10g to 100g per liter, further ballast nonalbumin proteins precipitate with ethanol, which are preferably removed by centrifugation, and then ethanol from the solution precipitates albumin in the form of Cohn fraction V, separation into aqueous solution and further processed. Process according to claim 1, characterized in that the precipitation of the ballast nonalbumin proteins is carried out with ethanol to a total concentration of 40 + 10% ethanol from the total volume of the reaction mixture at pH 5.8 + 0.3, at a final temperature of -5 ° C + 0.5 ° C, wherein the ballast proteins precipitate in the form of a fine suspension which is stirred at said temperature, preferably for 3 to 12 hours, then separated preferably by centrifugation and the resulting supernatant is subjected to a black filtration, preferably with diatomaceous earth. The method of claim 1, wherein the precipitation of albumin is performed from the supernatant after filtration with ethanol to a total concentration of 40 + 10% ethanol of the total volume of the reaction mixture at pH 4.8 + 0.3, at -14 ° C. ° C + 4 ° C, wherein the albumin precipitates in the form of a fine suspension, which is stirred at said temperature, preferably for 3 to 12 hours, then separated preferably by centrifugation, and the obtained albumin paste fraction V is subjected to further processing. 4. The method of claim 1 wherein the protein material of the albumin paste fraction V is dissolved in an amount of pyrogen-free water at a temperature of 0 ° C to 6 ° C such that the protein concentration is in the range of 10 g to 150 g liter at a pH in the range of 4.0 to 8.0, subjected to a black filtering, deprived of low-molecular substances by diafiltration or ultrafiltration, and the solution thus obtained is subjected to further processing.