RU2340356C2 - Method of obtaining of anthracic protective antigen - Google Patents

Abstract

FIELD: medicine; biotechnologies.

SUBSTANCE: plating of the vaccine strain on a sterile nutrient medium with sodium bicarbonate. Thus administer sodium bicarbonate into a nutrient medium throughout an exponential growth phase at 6-8 hours of the vaccine strain planting. After the planting process termination allocation, concentration and sterilisation of a protective antigen is performed on the equipment of ultra-and microstraining.

EFFECT: stable obtaining of a native anthracic protective antigen with the activity in "РДП" more that 100 EA·ml^-1 and reduction of time and stages of reception of the sterile concentrated protective antigen with the activity in "РДП" more that 200 EA·ml^-1.

3 tbl, 2 ex

Description

The invention relates to the microbiological industry, in particular to a technology for producing anthrax protective antigen (PA), in particular to anthrax vaccines, and can be used in medicine and veterinary medicine for specific prophylaxis of anthrax.

EFFECT: invention allows reproducibly producing native PA with activity in the diffusion precipitation (RDP) reaction in a gel of more than 100 EA · ml ^-1 , reducing the time and number of technological stages of obtaining sterile concentrated PA.

Anthrax PA is used to produce two types of vaccines: anthrax purified, adsorbed chemical and combined liquid and dry (one of the components of which it is), according to immunological and physico-chemical parameters that meet the requirements of the national body for the control of medical immunobiological preparations (MIBP) to these vaccines.

A known method of producing anthrax PA in deep anaerobic cultivation of B.antracis strain on a nutrient medium containing sodium bicarbonate during aeration of the culture fluid with an inert gas (patent RU 2223114 C2, 02/10/2004). This method is used in the preparation of a vaccine anthrax purified adsorbed chemical liquid, which includes the following stages:

preparation of the uterine seed culture of the production strain STI-1;

preparation of a production medium;

obtaining a native culture of strain STI-1;

obtaining native PA;

obtaining sterile PA, including the separation of native PA on a separator SA-1-02-575, concentration on a UFU-4.0 unit, coarse, fine and sterilizing filtration through a blue ribbon paper filter, MFA No. 2 and MFA No. 1 filter membranes ;

sorption of sterile concentrated PA on an aluminum hydroxide gel;

stabilization with formalin solution and washing the adsorbed PA;

dilution of sorbed concentrated PA, bottling;

quality control of the obtained sorbed concentrated PA.

The disadvantage of the above method is the low reproducibility upon receipt of native PA in fermenters with the required PA activity in the diffusion precipitation reaction of 100 EA · ml ^-1 or more. In addition, when separating PA from the culture fluid, the separation method is used, which is longer in time and has the following disadvantages (Research report. Improving the production technology of chemical anthrax vaccine - Arch. Vch 23527 - Inv. No. 30353, 1997. - P.28 ):

the complexity of operation (vibration, noise, the need for disassembly and washing) of the apparatus;

impact on the microbial culture of centrifugal force, differential pressure and hydrodynamic shock;

lack of sealing and ensuring aseptic conditions of the process.

In addition, it is known that when the microbial population of the vaccine anthrax strain STI-1 is grown in the presence of a bicarbonate ion added at the time of cultivation, the proportion of cells with structural breakdown of the pag gene under conditions of unbalanced growth grows (Sirard JC, Mocr M., Fonet A Tne three Bacillus anthracis taxin genesare coordinately regulated by bicarbonate and temperature. / J. Bacterid / - 1962 / - vol. 52, p.632-645), which negatively affects the production of antigen.

The objective of the invention is the stable production of native anthrax PA with high activity, reducing the time to obtain sterile concentrated PA with high activity.

The problem is solved by changing the moment of introduction of a solution of sodium bicarbonate during cultivation and obtaining sterile concentrated PA by ultrafiltration and microfiltration.

The possibility of practical use of the claimed invention is confirmed by the following examples.

Example 1. Changing the time of introduction of the inducer of synthesis of PA-solution of sodium bicarbonate (NaHCO ₃ ).

It is known that when the microbial population of the vaccine anthrax strain STI-1 is grown in the presence of a bicarbonate ion added at the moment of cultivation, the proportion of cells with structural breakdowns of the pag gene under conditions of unbalanced growth increases (Sirard JC, Mocr M., Fonet A. Tne three Bacillus anthracis taxin genesare coordinately regulated by bicarbonate and temperature. / J. Bacterid / - 1962 / - vol. 52, p. 632-645).

To reduce the negative impact on the development of microbial culture in the initial phase of growth caused by the presence of sodium bicarbonate in PS, it is advisable to introduce an inducer (NaHCO ₃ ) of PA synthesis in the dynamics of cultivation for 6 ... 8 hours of growth, which is controlled by the glucose content in the medium and the pH value culture fluid. Sodium bicarbonate activates the expression of the rad gene that determines PA synthesis in the late lag phase and during the exponential growth phase.

The dynamics of the development of the microbial population of the vaccine anthrax strain and the accumulation of PA according to the existing method (based on five cycles of fermentation) of the technology are presented in table 1, according to the claimed method in table 2 (based on 10 cycles of fermentation).

As can be seen from the results presented in tables 1 and 2, the introduction of sodium bicarbonate at 6 hours of cultivation according to the claimed method of technology, in contrast to the existing method, favorably affects the production of PA, the antigenic activity of which by 12 hours of cultivation reaches a regulated rate of 100 EA · ml ^{- 1} , and by 19 hours of growth, antigenic activity increases to 200 EA · ml ^-1 and remains at this level until the end of the cultivation process.

Example 2. The choice of effective equipment in the process of obtaining sterile concentrated PA.

The use of the Sartocon ultra- and microfiltration unit in the technological process of separation, sterilization and concentration of PA makes it possible to:

carry out the process in aseptic conditions;

sterilize equipment by chemical and thermal methods and their combination, which meets the requirements of GMP;

quickly replace filter elements in case of failure during the process due to the Sartokon modular filter system.

The inventive method of obtaining a sterile concentrated PA using the Sartocon installation during the micro- and ultrafiltration process reduces the number of stages from 6 to 2, reduces the time for the release of PA from the culture fluid and the whole cycle as compared to the existing method and includes:

the allocation of PA from the culture fluid and its simultaneous sterilization;

concentration of sterile PA.

The technological characteristics of the process of concentration of PA on the existing and claimed methods of technology are presented in table 3.

As can be seen from the data presented in table 3, the use of micro- and ultrafiltration unit "Sartocon" according to the claimed method significantly reduces the time for the release of PA from the culture fluid and the whole process as a whole compared with the existing method of technology.

Table 1 The dynamics of the development of microbial culture by the existing method, n = 5 The determined indicator Growing time, h 0 9 12 fifteen eighteen twenty pH units pH 8.4 8.1 7.7 7.6 7.4 7.0 Biomass, units the ex. 60 250 280 350 560 1300 Utilization of glucose,% 0 5 eighteen 28 43 68 Antigenic activity in RDP, EA. ml ^-1 0 25 25 25 ... 50 25 ... 50 25 ... 100

table 2 The dynamics of the development of microbial culture by the present method, n = 10 The determined indicator Growing time, h 0 6 12 19 pH units pH 7.7 7.2 / 7.9 * 7.6 7.4 Biomass, units the ex. 75 650 1480 1860 Utilization of glucose,% 0 16 79 one hundred Antigenic activity in RDP, EA. ml ^-1 0 0 50 ... 100 100 ... 200 Note: * - pH value of the culture before and after making sodium bicarbonate.

Table 3 Temporary characteristics of the technological process according to the existing and claimed method, n = 3 The stages of the technological process according to the existing method The time of the process according to the existing method, min The stages of the technological process according to the claimed method The time of the process according to the claimed method, min Isolation of PA from the culture fluid by separation using Westfalia SA-02-575 90 ± 10 Isolation of PA from the native culture and its simultaneous sterilization by microfiltration at the Sartakon-2 installation 40 ± 10 Concentration of native PA in the UFS-0.4 installation 90 ± 20 Concentration of sterile PA by ultrafiltration on a Sartacon-2 unit. 40 ± 10 Coarse filtration (Bunsen flask) 120 ± 10 absent absent Fine filtration, filter MFA-A No. 2 60 ± 10 absent absent Sterilizing filtration, filter MFA-A No. 1 60 ± 10 absent absent The total time for the existing method is 420 ± 20 minutes The total time of the proposed method is 80 ± 10 minutes

Claims (1)

A method of producing anthrax protective antigen, including culturing a vaccine strain on a sterile nutrient medium with sodium bicarbonate, characterized in that sodium bicarbonate is introduced into the nutrient medium during the exponential growth phase for 6-8 hours of cultivation of the vaccine strain, and after the cultivation process is completed, isolation, concentration and sterilization of the protective antigen is carried out in one ultrafiltration and microfiltration unit.