Method of obtaining of anthracic protective antigen

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.

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

The invention allows to reproducibly obtain native PA activity in the reaction diffusion precipitation (RDP) in the gel more than 100 EA·ml^-1to reduce the time and number of process steps of obtaining sterile concentrated PA.

Anthrax PA is used to obtain two types of vaccines: anthrax purified adsorbed chemical and combination of liquid and dry (one of the components which it is), immunological and physicochemical parameters that satisfy the requirements of the national authority of control of medical immunobiological preparations (mibp) to these vaccines.

A method of obtaining anthrax PA at deep anaerobic cultivation of strain B.antracis on a nutrient medium containing sodium bicarbonate when aeration of the culture fluid with an inert gas(patent RU 2223114 C2, 10.02.2004). This method is used in the preparation of anthrax vaccine adsorbed purified chemical liquid, comprising the following the steps:

preparation of master seed culture production strain STI-1;

preparation of production nutrient medium;

getting native culture strain STI-1;

getting native PA;

obtaining sterile PA, including the separation of native PA separator SA-1-02-575, concentrating on the installation UFA-4,0, gross, subtle, and sterilizing filtration through a filter paper of "blue ribbon", the filter membrane IFA # 2 and IPA No. 1;

sorption sterile concentrated PA gel aluminium hydroxide;

stabilization formalin solution and washing sorbed PA;

breeding sorbed concentrated PA, bottling;

the quality control of the obtained sorbed concentrated PA.

The disadvantage of the above method is the low reproducibility in obtaining native PA in fermenters with desired activity of PA in the reaction diffusion precipitation 100 EA·ml^-1and more. In addition, when the allocation of PA from the culture fluid used method of separation that time longer and has the following disadvantages (human development Report. Improving the technology of production of chemical anthrax vaccine Architect. RF 23527 - inv. No. 30353, 1997. - P.28):

the complexity of the operation (vibration, noise, the necessity of disassembly and cleaning) system;

effects on microbial culture centrifugal force, differential pressure and hydrodynamic blow;

the lack of sealing and ensure aseptic process conditions.

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

Object of the invention is stable reception of native anthrax PA with high activity, reducing the time of obtaining sterile concentrated PA with high activity.

The problem is solved by changing the moment of introduction of the sodium bicarbonate solution in the process of cultivating and receiving the sterile concentrated PA method for ultra - and microfiltration.

The possibility of practical use of the claimed invention, the following examples.

Example 1. Change the time of the introduction of the inducer of the synthesis of PA-solution of sodium bicarbonate (NaHCO₃).

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

To reduce the negative impact on the development of microbial culture in an initial growth phase, caused by the presence in the PS of sodium bicarbonate, it is advisable to introduce inductor (NaHCO₃synthesis PA in the dynamics of cultivation on 6...8 hours of growth, which is controlled by the concentration of glucose in the medium and the pH value of the culture fluid. Sodium bicarbonate activates gene expression happy determining the synthesis of PA, late lag phase and during the exponential phase of growth.

Dynamics of microbial populations anthrax vaccine strain and accumulation of PA on the existing method (based on five cycles of fermentation technology are presented in table 1, the claimed method in table 2 (based on 10 cycles of fermentation).

As can be seen from the results shown in tables 1 and 2, the introduction of sodium bicarbonate in 6 hours of cultivation by the present method technologies in contrast to the existing method has a positive effect on the production of PA, antigenic activity of which 12 hours of cultivation goes on regulated show the eh 100 EA· ml^-1and to 19 hours growth antigenic activity increases up to 200 EA·ml^-1and remains at this level until the end of the cultivation process.

Example 2. Selection of efficient equipment in the process of obtaining sterile concentrated PA.

The use of ultra - and microfiltration installation "Cartoon" in the process of selection, sterilization and concentration of PA allows to:

to conduct the process under aseptic conditions;

to carry out sterilization equipment chemical and thermal methods and their combination that meets the requirements of GMP;

quickly replace filter elements in case of failure during the process thanks to the modular filter system installation "Sartocon".

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

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

the concentration of sterile PA.

Technological characteristics of the process of concentri the Finance PA on existing and declared the ways technologies is presented in table 3.

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

The method of obtaining the anthrax protective antigen, including the cultivation of vaccine strain of sterile nutrient medium with sodium bicarbonate, wherein the sodium bicarbonate is lead into the nutrient medium during the exponential phase of growth at 6-8 h of cultivation vaccine strain, and after the end of the cultivation process selection, concentration and sterilization protective antigen carried out on a single installation of ultra - and microfiltration.