RU2451522C1 - Method for concentration of native cholerogen-anatoxin and o-antigen of 569 v serovar inaba strain classic biovar vibrio cholerae 01 - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: according to the invention, the method envisages preliminary concentration of native cholerogen-anatoxin and O-antigen produced in the course of submerged cultivation of cholera vibrio 569 V Inaba serovar strain and separated from the biomass by way of centrifugation performed on porous membranes in cross flow filtration mode. Concentration is performed through membrane with nominal molecular weight cut-off equal to 50 kDa in cross flow filtration mode under a pressure of 0.23-0.25 MPa, the average specific rate of ballast impurities from native cholerogen-anatoxin and O-antigen being 60-70 dm³/m²/h, the repetition factor of volumetric concentration of the material being filtered equal to 10. Then the ballast fractions are twice sediment with ammonium sulphate.

EFFECT: method usage allows to increase the yield of cholerogen-anatoxin and O-antigen by a factor of 1,2 and to decrease the quantity of ammonium sulphate used for ballast fractions sedimentation tenfold.

3 tbl

Description

The invention relates to a technology for the production of medical immunobiological preparations, in particular to methods for concentrating cholerogen-toxoid and O-antigen Vibrio cholerae O1 of the classic biovar strain 569 B of Inov serovar, and can be used in the practice of manufacturing an oral cholera bivalent chemical tablet vaccine.

Cholerogen-toxoid and O. antigen of V. cholerae O1 of classical biovar strain 569 B of serovar Inaba, along with O-antigen of V. cholerae O1 of classical biovar strain M-41 of serovar Ogawa, are the main components of the oral cholera bivalent chemical tablet vaccine.

This vaccine in 2001 by Order of the Ministry of Health of Russia (No. 229 dated 06/27/2001) was included in the National Calendar of Preventive Vaccinations for epidemiological indications, providing for vaccination of people traveling to countries that are not at home by cholera and the population of the border regions of Russia in case of adverse on cholera situation in the adjacent territory.

A known method of producing purified cholera O-antigen (Russian patent No. 2143280, class А61К 39/106, 1999), the essence of which is that ballast proteins are removed from the native O-antigen by precipitation at pH 4.4 ± 0 , 2, then non-specific impurities with a molecular weight of less than 100 kDa are concentrated and separated by ultrafiltration, and non-specific impurities with a molecular weight of less than 300 kDa are separated by column chromatography.

However, the O-antigen isolated by this method can be used only for specific indication and diagnosis of cholera, and is not used as one of the components of the vaccine. In addition, in the preparation obtained by this method, one of the components of the bivalent chemical tablet cholera vaccine, namely cholerogen-toxoid, is missing.

A known method of producing purified cholera exotoxin (V.V. Stroganov and others. The use of ultrafiltration method on domestic semipermeable membranes of the UAM brand for separating somatic O-antigen from exotoxin cholera vibrio - Probl. Especially dangerous inf., 1978, issue 4, p. 52-55) the essence of which is that from the native culture containing O-antigen and cholera exotoxin, O-antigen is removed by ultrafiltration.

The resulting cholera exotoxin is used only to obtain anticholerogenic serum, and not as a component of the vaccine. In addition, in the preparation obtained by this method, one of the components of the bivalent chemical tablet cholera vaccine is missing, namely the O-antigen.

A known method for the production of a chemical vaccine consisting of cholerogen-toxoid together with O-antigen of Serovar Inaba is described in Russian patent No. 2076734, A61K 39/00. Concentration consists in sequential fractionation of ammonium sulfate from the culture fluid of the Inaba strain of the Inaba somatic O-antigen between 0.2 and 0.35 saturation and the toxoid cholerogen between 0.35-0.8 saturation.

Known and closest to the claimed solution is a method of concentration of cholerogen-toxoid and O-antigen, described in the industrial regulations for the production of vaccine cholera bivalent chemical tablet No. PR 01898109-05-06 (RosNIPCHI "Microbe", Saratov, 2006) , the essence of which is that the native cholerogen-toxoid and O-antigen obtained by deep cultivation of V. cholerae O1 of the classic biovar strain 569 V of Inaba serovar and separated from the biomass by centrifugation (microbial centrifugate) concentrate ruyut using a number of sequentially successive operations: deposition ballast fraction by adding 281 ± 7,6 g of ammonium sulfate per 1 dm ³ germfree centrifuged followed by removal of this fraction by centrifugation; precipitation of cholerogen-toxoid and O-antigen from the obtained centrifugate by adding 327 ± 38 g of ammonium sulfate per 1 dm ^{3 of a} microbial centrifugate, followed by their concentration by centrifugation. The disadvantage of this method is the significant loss of cholerogen-toxoid and O-antigen and the use of a significant (608 ± 45.7 g per 1 dm ³ microbial centrifugate) amount of ammonium sulfate spent on deposition.

The technical result consists in increasing the yield of the products obtained - cholerogen-toxoid and O-antigen, while maintaining the quality indicators of the drug. The economic effect of using the invention is to reduce the cost of the final product.

To solve this problem, an additional technological operation was introduced: preliminary concentration of a 10-fold microbial centrifugal by flow filtration (cross-flow) through membrane modules with a nominal cut-off of a molecular mass of 50 kDa under a pressure of 0.23-0.25 MPa with an average specific release rate native choleragen-toxoid and O-antigen of ballast admixtures - 60-70 dm ³ / m ² / hr, followed by precipitation by adding ballast fraction 281 ± 7,6 g of ammonium sulfate per 1 dm ³ germfree with centrifugate osleduyuschim removal of this fraction by centrifugation; the precipitation of cholerogen-toxoid and O-antigen from the obtained centrifugate by adding 327 ± 38 g of ammonium sulfate per 1 DM ³ microbial centrifugate and concentration by centrifugation. Comparison of the essential features of the proposed method for producing concentrated cholerogen-toxoid and O-antigen show that the process of separating ballast impurities and the concentration of cholerogen-toxoid and O-antigen are common to them.

An essential distinguishing feature of the proposed method is the introduction of an additional technological operation — separation of ballast impurities through membrane filters with a nominal cut-off with a molecular weight of 50 kDa, which allows to increase the yield of the final product and reduce its cost by reducing the amount of microbial centrifugate subjected to precipitation with ammonium sulfate.

The possibility of carrying out the claimed invention is confirmed by the following example.

Example. Obtaining concentrated cholerogen-toxoid and O-antigen of V. cholerae O1 of the classic biovar strain 569 B of Inov serovar with an increase in the yield of the obtained product and a decrease in its cost, while maintaining the quality indicators of the drug.

We used native cholerogen-toxoid and O-antigen V.cholerae O1 of the classical biovar strain 569 B of Inov serovar obtained by deep cultivation and separated from the biomass by centrifugation according to production schedule No. PR 01898109-05-06. The amount of the microbial centrifugate of strain 569 B V. cholerae O1 of the classic biovar of Serovar Inab for producing cholerogen-toxoid and O-antigen according to the prototype method and the claimed method was 10.0 dm ³ . For preconcentration of native cholerogen-toxoid and O-antigen, membrane modules with a nominal cutoff of 50 kDa molecular weight were used.

Concentrated cholerogen-toxoid and O-antigen were obtained by recirculation through a system (installation with a cassette module) with the filtrate draining into a separate container, followed by precipitation of the ballast fraction by adding 281 ± 7.6 g of ammonium sulfate per 1 dm ^{3 of a} microbial centrifugate, followed by removal this fraction by centrifugation; the precipitation of cholerogen-toxoid and O-antigen from the obtained centrifugate by adding 327 ± 38 g of ammonium sulfate per 1 DM ³ microbial centrifugate and concentration by centrifugation. To conduct the process, experimentally selected technological parameters, which are presented in table 1.

From the data given in table 1, it follows that the experimentally selected parameters allow, at pressure values of 0.23-0.25 MPa, to achieve the optimal average specific rate of release of native cholerogen-toxoid and O-antigen from ballast impurities - 60-70 dm ³ / m ² / h The ratio of the concentration of the filtered material by volume is 10, which allows to reduce the amount of ammonium sulfate by 10 times in the proposed method. In this example, the amount of ammonium sulfate spent on precipitation by the prototype method was 6080 g, and by the claimed method 608 g. The calculation of the cost of consumables for obtaining a series of cholerogen-toxoid and O-antigen from 10.0 dm ^{3 of a} microbial centrifuge with using the prototype method and the proposed method, are shown in table 2, shows that the economic effect of the proposed method is 273.78 rubles (in 2003 prices).

Characteristics of the preparations obtained using the prototype method and the proposed method are shown in table 3.

According to the data obtained, the yield of the obtained product (cholerogen-toxoid and O-antigen) when using the proposed method increases by 1.2 times. In addition, the data in table 3 indicate that the values of the indicators characterizing the drugs do not differ from each other, which indicates the preservation of quality indicators of the drug obtained using the proposed method.

Table 1 The method of concentration of native cholerogen-toxoid and O-antigen Pressure, MPa The rate of release of native cholerogen-toxoid and O-antigen from ballast impurities, dm ³ / m ² / h 0.14-0.16 20-30 0.2-0.22 40-50 0.23-0.25 60-70 0.26-0.28 40-50

table 2 The method of concentration of native cholerogen-toxoid and O-antigen Consumables List Quantity per lot Cost per unit, rubles Cost per series, rubles Economic effect, rubles according to the prototype method by the claimed method according to the prototype method by the claimed method Ammonium sulfate, kg 6.08 0.608 50,0 304.0 30,4 Electricity, kW / h 3172.6 3172.6 1.96 7603.44 7603.44 Saturated steam, kg 320 320 0.67 214.4 214.4 Household water - drinking, m ³ 16.52 18.32 1,50 24.66 27.48 Compressed air, m ³ 4.4 4.4 0.98 4.30 4.30 Total: 8150.8 7877.02 273.78

Table 3 Name of indicator The value of the quality indicator of the drug obtained by the prototype method obtained by the claimed method Specific harmlessness harmless Specific activity of cholerogen-toxoid, toxoid binding units 6000 6000 The content of O-antigen, the inverse of the titer in the reaction of indirect agglutination with "O1" serum 256 256 The amount of product, g 2.5 3.0

Claims (1)

The method of concentration of native cholerogen-toxoid and O-antigen of Vibrio cholerae O1 of the classic biovar strain 569 B of Serovar Inaba in the production of a bivalent chemical tablet cholera vaccine, including sequential double precipitation of the first ballast fraction and then cholerogen-toxoid and O-antigen followed by ammonium sulfate characterized in that the native cholerogen-toxoid and O-antigen obtained by deep cultivation of V. cholerae O1 classic biovar strain 569 In the serovar Inaba and about separated from the biomass by centrifugation, they are pre-concentrated 10 times through membranes with a nominal cutoff of a molecular weight of 50 kDa in a cross-flow filtration mode with a pressure of 0.23-0.25 MPa, average specific release rate of native cholerogen-toxoid and O -antigen from ballast impurities - 60-70 dm ³ / m ² / h.