The method of producing dextran

 

(57) Abstract:

Usage: biotechnology, chemical technology, medical industry to obtain a drug of low molecular weight dextran. The inventive method of obtaining dextrans with srednevekovoi molecular weight of 1000 Days In the way that partial cleavage of high molecular weight dextran by acid hydrolysis in aqueous solution in the presence of hydrochloric acid by heating the acidified aqueous solution of high molecular weight dextran is passed through celinesite cationite ion-exchange resin macroporous structure on the basis of sulfotyrosine copolymers of styrene with divinylbenzene in the H+form, having a pH of 0.85 to 1.1 at a temperature 851oC with a speed of 0.01 to 0.25 volume per hour.

The invention relates to the field of biotechnology and chemical technology, particularly to a method of depolymerization of dextrans, namely the method of production of dextran from srednevekovoi molecular weight (m m) 1000 D, which can be used in the medical industry, the drug is able to prevent severe anaphylactic reactions during transfusion dextranomer products with higher m is a dextran with srednevekovoi m m 1000 D. It is produced by enzymatic synthesis of native dextran from sucrose using microorganisms of the species. Next native dextran is undergoing a profound hydrolysis and fractionation to obtain a product with the required m m (1).

Specified drug has the properties of monovalent hapten. The substance of dextran with m 1000 m D react only with the individual reaction center dextranomer antibodies without the formation of the next "bridge" connections and has no tendency to the formation of immune complexes.

An excess of monovalent hapten, administered by intravenous influence, competes with polyvalent haptens (dextran drugs with higher m m) in the reaction of formation of immune complexes and prevents anaphylaxis.

Widely known method of partial cleavage of native dextran, which is carried out by acid hydrolysis (2). The process is carried out in acidic medium at temperature 85 87oC and constant stirring. This method is used to produce dextrans with m m and 40000 70000 D. the Disadvantage of this method is the impossibility of obtaining low molecular weight dextrans with the desired molecular weight, since the deep is available dextran molecules with small m m and as a result significantly increases the proportion of di - and monosaccharides in the final product of hydrolysis.

This method aims to obtain dextran with a molecular weight of 1000 D.

For this purpose, the method of partial cleavage of high molecular weight dextran by acid hydrolysis in aqueous solution in the presence of hydrochloric acid by heating the acidified aqueous solution of high molecular weight dextran is passed through celinesite cationite ion-exchange resin macroporous structure on the basis of sulfotyrosine copolymers of styrene with divinylbenzene in the H+form, having a pH of 0.85 to 1.1 at a temperature 851oC, with a speed of 0.01 0.25 volume per hour.

The method in accordance with the invention is as follows: a glass column (height 600 mm, diameter 35 mm) is filled with cationite ion exchange resin (the resin 500 ml), passed through it sequentially 1 M NaOH solution, distilled water, 1 M hydrochloric acid and washed with distilled water to a pH of 0.85 to 1.1. An aqueous solution of high molecular weight dextran with a concentration of 5 to 10% acidified with hydrochloric acid (pH 1.0 to 1.5) and passed through a column of ion exchange resin with a speed of 0.01 0.25 volume per hour. The process of cartografia on Sephadex C-15, which records the velocity of the aqueous solution of the original dextran through the cation exchanger.

Get the solutions of partially hydrolyzed dextran with an average molecular weight of 1300 1700 D, which is subjected to fractionation on Sephadex C-15 on the column, calibrated with dextran 1000 with a speed of 150 - 220 ml per hour.

The advantage of this method is the possibility of deep hydrolysis of high molecular weight dextran to dextran with m m 1000 D with the release of the final product is about 50%

The proposed method allows to fully automate the process and implement the continuous flow method without the use of large quantities of inorganic acids, which significantly increases the technical-economic indicators of technology and solves the problem of ecology.

Use as a solid-phase catalyst ion-exchange resins with pore size from 250 to 2500 macroporous structure with highly developed specific surface (up to 350 m2/g), containing SO3H group, allows hydrolysis process with high speed and high selectivity molecular masses due to the high diffusion coefficients of molecules of dextran in macropores sledovatelno miss a 1 M solution WaOH, distilled water, 1 M hydrochloric acid and washed pyrogen-free water to a pH of 0.85, the system thermostatic at a temperature 851oC. an Aqueous solution of native dextran (C= 5%) acidified with hydrochloric acid to pH 1.5 and passed through the column and ion-exchange resin with a speed of 0.01 to 0.04 colonoscope volume per hour.

The resulting solution of partially hydrolyzed dextran portions of 200 ml fractionary on a column (diameter 40 mm, height 600 mm), filled with media Sephadex C-15 (700 ml), with a speed of 0.32 column volume per hour, taking the fraction corresponding to the time of output to the standard dextran 1000, and freeze-dried. The yield of dry matter 48% Mw=1200 D. Prepare a formulation containing 15% substance dextran and 0.6% sodium chloride solution, sterile filtered, bottled and sterilized in the usual way.

Get a sterile, pyrogen-free, clear, slightly yellow solution of the drug, not differing in physicochemical and biological properties of the drug Promit.

Example 2. Column, prepared as described above and washed pyrogen-free water to a pH of 0.96, thermostatic at a temperature 851oC. an Aqueous solution of dextran with m m 6000010000 D (C=10%) acidified solace and fractionary with the speed of 0.27 volume per hour as described above. The yield of dry matter 53% Mw=1000 D. Preparing a dosage form containing 14.7% of the substances of low molecular weight dextran and about 6 sodium chloride solution, sterile filtered, bottled and sterilized in the usual way.

Get a sterile, pyrogen-free, clear, colorless solution of the drug, not differing in physicochemical and biological properties from the Swedish drug Promit.

Example 3. Column, prepared as described above and washed until the pH of 1.1, thermostatic at a temperature 851oC. an Aqueous solution of dextran with m m 50001500 D (C= 8%) acidified with hydrochloric acid to pH 1.2 and passed through the column and ion-exchange resin with a speed 0,18 0,25 column volume per hour, fractionary with speed,20 column volume per hour as described above. The yield of dry matter 49% Mw=1100 D. Prepare a formulation containing 15% of a substance of low molecular weight dextran and 0.6% sodium chloride solution, sterile filtered, bottled and sterilized in the usual way.

Get a sterile, pyrogen-free, clear, colorless solution of the drug, not differing in physicochemical and biological properties from the Swedish drug Promit.

Way obtained the second acid when heated, wherein the acidified aqueous solution of high molecular weight dextran is passed through an ion-exchange resin based on a sulfonated copolymer of styrene with divinylbenzene in the N+form, having a pH of 0.85 to 1.1, with a speed of 0.01 to 0.25.h, the resulting hydrolysate is passed through a column Packed with the carrier of the Sephadex C-15, at a rate of 0.2 0,32 about./h, and the resulting fraction dextran (mol.m. 1000 D dried with known methods.

 

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