A method for concentrating spore cultures in the production of the anthrax vaccine |
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IPC classes for russian patent A method for concentrating spore cultures in the production of the anthrax vaccine (RU 2151798):
 Lactobacillus acidophilus k/ar-08 - antibiotic-resistant and salt-tolerant antagonist agents of intestinal infections / 2151797
The invention relates to biotechnology and medical Microbiology, may be used in medicine, biotechnology, upon receipt of probiotic bakpreparatov medical and milk products, nutritious diet food
The strain of lactococcus lactis producing the lowland bacteriocin / 2151796
The invention relates to biotechnology and represents a new strain Lactocoсcus lactis PMBC-In-7699 - producer of food polypeptide preservative nisin
Method for activating yeast / 2151794
The invention relates to the food industry and relates to fermented products
 The method of producing antibiotic actinomycin, strain streptОmyces (kitasatoa) species 834 arriam d-484 - producer of the antibiotic actinomycin / 2151793
The invention relates to biotechnology, in particular to the production of antibiotics
 The method of determining the effectiveness of steam sterilization bacteriological method / 2151187
The invention relates to medical and veterinary Microbiology and relates to a method of monitoring the effectiveness of sterilization of medical and veterinary use thermal method, namely sterilization heated with saturated steam
 The influenza virus strain a/47/johannesburg/96/7/7 (h1n1) for production of live intranasal influenza vaccine for children / 2151185
The invention relates to the production of vaccine strain influenza a/47/Johannesburg/96/7/7 (H1N1) for production of live intranasal influenza vaccine for children
 The strain of the fungus entomophthora thaxteriana n19-11 (vizr) to obtain biological mihaylin t / 2150836
The invention relates to the field of agricultural Microbiology, and can be used to obtain drug mihaylin T to protect plants from aphids, spider mites and thrips
 The strain of bacteria desulfovibrio vulgaris "east-5", used for the treatment of wastewater from heavy metal ions / 2150503
The invention relates to biotechnology and industrial wastewater treatment
 The strain of bacteria desulfovibrio baarsii "east-6", used for the treatment of wastewater from heavy metal ions / 2150502
The invention relates to biotechnology, industrial wastewater treatment
Bacterial strain vibrio cholerae 0139 of serovar km 115 (1262) , avirulent test strain not containing ctx ab operon, immunological and genetic studies / 2149898
The invention relates to the field of Microbiology, namely Microbiology cholera, and can be used in immunological and genetic studies as avirulent test 0139 strain of serovar not containing the ctx operon AB gene, determining the synthesis of cholera enterotoxin
A method of manufacturing a vaccine against anthrax in animals / 2095409
The invention relates to the field of Microbiology, in particular biotechnology, vaccines, and can be used in the manufacture of vaccines against anthrax
 The method of obtaining spores of microsporidia / 2049395
The invention relates to agriculture, in particular plant protection
 The method of receiving eubacteria / 2036232
The invention relates to biotechnology, in particular to the production of medical biological preparations
The method of producing antibiotic actinomycin, strain streptОmyces (kitasatoa) species 834 arriam d-484 - producer of the antibiotic actinomycin / 2151793
The invention relates to biotechnology, in particular to the production of antibiotics
Bacterial strain vibrio cholerae 0139 of serovar km 115 (1262) , avirulent test strain not containing ctx ab operon, immunological and genetic studies / 2149898
The invention relates to the field of Microbiology, namely Microbiology cholera, and can be used in immunological and genetic studies as avirulent test 0139 strain of serovar not containing the ctx operon AB gene, determining the synthesis of cholera enterotoxin
Biotechnological method of purification of components of the equipment from contamination / 2146293
The invention relates to the field of biotechnology, namely microbiological purification method from contamination (sludge, varnish deposits and other) aircraft and other components of the equipment in operation
Biosorbent / 2143318
The invention relates to the field of biotechnology, in particular to the waste disposal processes of the food industry, and can be used in the recycling process of microbiological production of ethyl alcohol
 The method of obtaining biologically active supplements in dry form containing bacteria-eubiotics, and filling for bread or confectionery products / 2142504
The invention relates to biotechnology and the food industry, namely, technologies for food additives
 A method of obtaining a sterile solution of trypsin for cell cultures / 2142503
The invention relates to biotechnology and relates to a method of obtaining sterile trypsin solution used to obtain primary, maintain diploid and transplantable cell lines
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(57) Abstract: The invention concerns the technology of production of medical immunobiological preparations. The concentration of spore suspensions in the manufacture of vaccines carry on porous membranes in the mode of flow (cross-flow) filtration. For the Department of cell biomass using membranes with a pore size of 0.2 μm. Concentration is carried out in the mode of flow filter with a pressure of 0.15-0.2 MPa and a speed of material flow 12-16 DM3h-1. The wash is a concentrated spore suspension spend distilled water in the ratio 1:3. The invention eliminates inactivation and reduce losses dispute to 10% by creating gentle conditions concentration of the dispute and to increase the degree of purification from ballast substances. table 2. The invention relates to the production technology of medical immunobiological preparations, in particular, to a method of concentrating spore cultures in the production of the anthrax vaccine, ensuring the stability of their biological properties with preservation of potency, and can be used in the practice of production of the anthrax vaccine preparatoire dispute. Sedimentation spore suspensions is using this technology for more than ten days at a temperature of 2-4oC, which is attributed to higher viscosity of the liquid phase, associated primarily with the presence of a protein substrate-product of the lysis of bacterial cells and nutrient medium components (NIELS, , Tbilisi, Georgia). This method has significant drawbacks: the process is conducted for a long time at low temperatures; drugs prepared on the basis of these concentrates have a high reactogenicity. The known method the concentration of bacterial spores (ed. the certificate of the USSR N 835170 class. 12 N 1/002, 1983), the essence of which is that in the spore suspension contribute polyethylenimine at a concentration of 0.05-0.1%. By introducing additives settling time is reduced to 4-5 days, the temperature during deposition is 1-15oC. This method, although increases the yield of microbial biomass, but at the same time creates difficulties in terms of its liberation from the precipitator. In conditions of industrial production of vaccines for people having precipitator in the vaccine is highly undesirable and usually worsens its quality characteristics. Closest to the claimed solution is a method for industrial preparation of spore concentrates for the preparation of the anthrax vaccine centrifugal method (regulation of production of live dry anthrax vaccine N 364-95, scientifically defense Ministry, G. Kirov, 1995). This method includes the following stages: technical training separators and metal air filters, heat sterilization of the separator, the primary concentration of cell biomass, clean ballast impurities sterile distilled water in a ratio of one to ten, secondary concentration of cell biomass, collecting the concentrated spore suspensions in sterile container, sterilization separator. In addition, in the preparation of the separator to work all detali machines perform halide leak detector GTI-6. Sterilization of the separator is carried out thermally. The vapor pressure of 0.05-0.06 MPa. Exposure time is 1.9-2.1 hours. To obtain the primary concentrate of the spore suspension to the fitting issuing from separator in compliance with the requirements of asepsis connect two containers of 20 DM3with 10 DM3sterile distilled water, and the supply line of the spore culture establish capacity with a capacity of 20 DM3with 10 DM3sterile distilled water. Then include a separator and serves spore culture in the cavity Boxing separator with a speed of 30-40 DM3h-1. In the process of separation in the bioreactor is maintained a pressure of 0.015-0.025 MPa. It should be noted, that created by the rotating liquid in the separator pressure on disputes corresponds to 2.0 to 2.5 MPa. After 50-60 minutes stop filing spore culture and from the container into the cavity of the box send 1,8-2,1 DM3sterile distilled water for cleaning primary spore concentrate. Then, wearing gloves, mounted on the box separator, disassemble the drum and using a special knife collecting spore pasta in a glass. The contents of the last resuspending in 0,6-1,2 DM3sterile distiller is in one of the containers with water, then disconnect it from the socket. Obtaining primary concentrate is up to 4 hours (volume of processed spore culture -80 DM3). Obtaining secondary concentrate. Prior to the fitting of filing spore culture connect tank with a capacity of 20 DM3with 10 DM3sterile distilled water and two containers washed the primary concentrate on the dispensing line of the spore suspension to the fitting mount glass bottle with a capacity of 5 DM3. Creating a pressure of 0.01 to 0.02 MPa in consumable containers, include the separator, to direct the flow of the primary concentrate in the drum separator. Volumetric flow rate is 10-12 DM3h-1. The stop of the separator is carried out after the processing of the first bottle with the primary concentrate. Re-suspension of the secondary concentrate perform as described above, after which the secondary concentrate spore suspension unloaded in a container with a capacity of 5 DM3. The second tank with the primary concentrate sephirot like the first. The collection of biomass produced in the tank that is installed on the dispensing line of the spore suspension. Cooking time secondary concentrate up to 3.5 hours. The total value of losses when receiving the Knossos dispute with centrate). Centrifugal method of obtaining a concentrated suspensions has the following disadvantages: low biomass yield of cells due to the loss; large energy and cost of manual labor; the difficulty of achieving aseptic process conditions. Increasing the feed rate of spore cultures in the drum of the separator increases the percentage of losses, while limiting the feed rate of loss is reduced due to the smaller ash dispute with the centrate, but it increases the duration of the process and, consequently, heating of the biomass. Exceeding regulatory time separation spore suspensions entails an increase in the values of the temperature of the spore concentrate in the parietal layer of the rotor of the centrifugal machine. So, when heating the concentrate within one hour to a temperature of 50oC the death of the dispute is 28%, while heating to 60oC is the death of 37% dispute. Design of centrifugal machines used in the production of the anthrax vaccine, allows you to maintain acceptable temperature 20-50oC not more than 6 hours. However, even taking into account the selected rational modes of concentration calculation of total material balance of the process polki spore concentrate when separation is necessary for removal of biomass from the ballast impurities and it is performed manually by an operator. This requires a large investment of time and manual labor. The task of the invention to provide a concentrated spore suspensions in aseptic conditions, characterized by increased biomass yield, increased degree of purification from ballast impurities and providing gentle conditions of concentration and the creation of highly immunogenic and erectogenic vaccines. To solve this task, the concentration was carried out by flow filtration through membrane modules with a pore size of 0.2 μm under pressure of 0.15-0.2 MPa with the speed of material flow 12-16 DM3h-1with the subsequent purification of cell biomass in a ratio of 1:3. A comparison of the important features of the proposed method of obtaining concentrated spore suspensions and prototype show that their common feature is the separation of cell biomass and its clearance from the ballast impurities with subsequent separation of the cellular biomass. Salient features of the proposed method are: Department of cell biomass through membrane filters with a pore size of 0.2 μm, to prevent inactivation and reduce losses to dispute ü material flow 12-16 DM3h-1when the primary and secondary concentration can increase the processing capacity of spore culture from 80 to 120 DM3due to the high permeability of the membrane, providing a large volumetric flow of the product; the proposed hydrodynamic pressure of 0.15-0.2 MPa can significantly reduce its adverse impact on disputes by eliminating the influence of centrifugal forces of the rotating speed 9000 rpm drum separator;the volume ratio of concentrated spore suspensions with distilled water 1: 3 allows users to more efficiently perform the process of washing from the ballast impurities by maintaining in liquid homogeneous condition spore concentrate that can largely be cleansed of impurities, which is characterized by the content of total nitrogen. This object is achieved by the choice of parameters of technological process on porous membranes and conditions ensuring fold lower loss, sterility concentrated preparations and stability of their biological properties. Obtaining a concentrated spore suspension on a porous membrane includes a t is dy, and check the installation for leaks, its sterilization and obtaining a concentrated spore suspension. To solve the task it was applied membrane modules with a pore size of 0.2 μm. Cassette membrane module is a rectangular closed unit. It contains a package of filters arranged in parallel, between which there is a layer of tissue substrate. The original thread spore culture moves inside of the substrate in the narrow gap between the pairs of membranes. The substrate of tissue causes turbulence in the filtered liquid, which counteract the formation of the blocking layer and strengthen the effect of tangential flow direction applied to the membrane. Each module on the upper and lower edge provided with a number of holes that align with corresponding holes of the filter holder installation for organizing the flow of fluid. Holes are made in the front and rear clamping plates. The filtered liquid is supplied through five openings on the bottom edge of the substrate between the filters. The filtrate is discharged through the relevant final five holes on the top edge of the module. The concentrate passes through four remaining holes on Vertolet to avoid inactivation of biological products, virtually eliminated the concentration polarization. The small dead volume of product loss to a minimum. Membrane modules are sterilized in both thermal and chemical methods. The concentration process is carried out under aseptic conditions. The essence of the proposed method is to obtain a concentrated spore suspensions in the manufacture of vaccines on porous membranes in the mode of flow (cross-flow) filtering. Enablement of the claimed invention, the following examples. Example 1. Obtaining a concentrated spore suspensions of membrane method with increased biomass yield, increased degree of purification and high immunogenicity. For used spore culture Bacillus anthracis vaccine strain STI-1, obtained by the method of deep cultivation according to the rules PR-364-95. For concentration spore cultures have used 1 - 5 modules "Microsort on the basis of polyolefin with a pore size of 0.2 μm (the diameter of pores of the membrane were chosen based on the size of Bacillus anthracis) and the area of the filter surface 0.6 m3. From the data in table 1 it follows that experimentally chosen parameters allow the pressures of 0.15-0.2 MPa to achieve optimum feed speed of the spore suspension - 12-16 DM3h-1. The duration of the process when the concentration of volume 120 DM3the spore suspension is 6.5-7.5 hours, while the temperature of the spore concentrate does not exceed 25oC. the Ratio of the concentration ratio of the filtered material by volume equal 8-14 times, which corresponds to the centrifugal method of separation while reducing losses spore biomass with 50% when centrifugal, up to 8-10% in the proposed method. Characteristics of spore-bearing concentrates derived membrane as described in table 2. These tables indicate that the values of the final concentration of spores in the drugs exceed the performance of the prototype in average two times. All other indicators spore concentrates comply with the requirements of the rules of live dry anthrax vaccine. It should be noted that in the vaccine cost samples vaccines, received on the proposed technology fully meets the requirements of FS 42-3269-96. The absence of extraneous microflora confirms the correctness of the choice of sterilization method, type of disinfectant, operation, cleaning, and maintenance of technological processes in aseptic conditions with the proposed technological regime. Example 2. Ensure aseptic conditions. It was established experimentally that the application of 2-3% solution of formalin for the current and post-processing microfiltration plants and cassette modules, and also as a preservative membrane modules with long breaks in operation, storage and transportation, provides, in combination with a hermetic design microfiltration equipment, sterility obtained spore concentrates. This was experimentally proved the following modes of sterilization: chemical sterilization by recirculation of 2-3% formalin solution for at least 40 minutes; the cleaning system from the remnants of the disinfectant 15-20 DM3sterile distilled water. After the operation of washing with sterile distilled water quantitative ostavlaet from 0.001 to 0.007%, that meets the requirements of the Ministry of health of the USSR No. 31 dated January 13, 1981, applicable to this kind of medical immunobiological preparations. Example 3. The concentration of low-yielding spore suspensions. For objective evaluation of the potential of membrane concentration method used low-yielding (from 0.5 to 1.0 billion spores. cm-1) spore culture. When using low-yielding spore suspensions concentration of spores upon completion of the process of microfiltration reached 12-6 billion cm-3that, like other indicators, satisfies regulatory requirements in the production of the anthrax vaccine. This not only indicates a high concentrating ability of the method and optimality selected values of technological parameters of the process, but also allows for the production of vaccines low yielding spore culture - spec for centrifugal concentration method according to regulations PR-364-95. Economic efficiency and environmental safety. An important advantage of membrane technology in comparison with centrifugal machines is their relatively low stimulator "Westphalia (Germany) is one million two hundred roubles, and the membrane unit "Sartocon-2" (Russia) with a set of membrane modules and tooling is a hundred and twenty thousand rubles (in prices of 1998). With virtually no capital costs for creating the technological stage of concentration in the production of the anthrax vaccine. The use of chemical sterilization for current and post-processing does not require the use of steam, which significantly reduces the energy intensity of production. A distinctive feature of the proposed method concentration is no aerotolerant biological products at concentration due to the tightness of the installation of microfiltration. Hermetic performance installation and low values of hydrodynamic pressure require the development and creation of the Boxing devices to protect the environment and personnel from the effects of processed biological products. A method for concentrating spore cultures in the production of the anthrax vaccine, including the Department of cell biomass, its clean ballast impurities with subsequent separation of the cellular biomass, characterized in that the concentration of the wasp is the material flow 12 - 16 DM3h-1and cleaned with distilled water in the ratio 1 : 3.
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