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Method of obtaining preparation based on vaccine strain of plague microbe. RU patent 2510825.

Method of obtaining preparation based on vaccine strain of plague microbe. RU patent 2510825.
IPC classes for russian patent Method of obtaining preparation based on vaccine strain of plague microbe. RU patent 2510825. (RU 2510825):

C12N1/20 - Bacteria; Culture media therefor
C12N1/04 - Preserving or maintaining viable micro-organisms (immobilised micro-organisms C12N0011000000)
A61K39/02 - Bacterial antigens
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FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with method of obtaining preparation based on vaccine strain of plague microbe. Claimed invention includes preparing inoculation native culture of plague microbe, concentration of microbe suspension, preparing vaccine suspension and obtaining dry form of preparation, with process of preparing inoculation culture including cultivation of microbes in liquid nutritional medium in flasks for 48 h at temperature 26…28°C and contibuous aeration with not less than 10 l min-1. with passaged stabilised starting culture, obtained as a result of three successive passages through organism of guinea pigs and mixed with glycerol-lactose-polyglucinum liquid in ratio 2:1; for preparation of vaccine suspension used is optimised in component composition protective drying medium, lyophilisation being carried out with observance of the specified regimen.

EFFECT: claimed solution makes it possible to obtain product with higher activity with reduced duration of process of its manufacturing.

3 dwg, 6 tbl

 

The invention refers to biotechnology, namely to methods for bacterial preparations based vaccine strains of the plague microbe, and can be used for preparation of medical immunobiological preparations.

Currently in Russia only drug used for specific prophylaxis of plague is the plague vaccine live dry, developed in 1937...1939 on the basis of the vaccine strain EV line NIIEG [Fibich MM, Karneev R.V., 1947]. Prolonged use of the vaccine plague living dry showed its high efficiency in cutaneous, intradermal, subcutaneous and inhalation ways of immunization.

However, the problem of preservation of highly immunogenic properties of the vaccine is largely determined by the stability of the biological properties of vaccine strains of the plague microbe, included in its composition, not only in the process of long-term storage, but also on the technological stage of production of the drug. Produced a series of vaccines, as practice shows, may vary according to the physical-chemical properties, the content of live microbial cells, thermal stability, residual moisture, immunogenicity and other indicators. Therefore, the search for new ways of getting plague vaccines, improving their qualitative characteristics, is an important task at the present stage.

A method of obtaining vaccine plague living dry from the strain of the EV line NIIEG used in NIPCI, Stavropol [Industrial regulations. Registration number fgun gisk named after. L.A. Tarasevich IR # 2334-09]. The method consists in sequential preparation of production of freeze-dried culture vaccine strain EV crop...I III generation: I generation is growing in test tubes on the sloping agar of Hottinger, II generation - in mattresses (bottles) with the broth of Hottinger or broth-based enzymatically hydrolyzed casein (PGA), III generation - cultivation in liquid nutrient medium (HRSA) in bottles, the cultivation of native culture on a nutrient dense environment (PPP) in AKM-W (reactor), cooking vaccine suspension and dry product.

Closest to the claimed is a way of getting the vaccine plague living dry-based vaccine strain EV used in 48 Central research Institute of defense Ministry of Russia [Industrial regulations on production of a vaccine plague live freeze-dried preparation suspension for injections, inhalation and skin skarifikih application. PR 08461522-07-08. Approved 03.06.2009, Registration number fgun gisk named after. L.A. Tarasevich IR # 2120-09]. The method consists in sequential preparation of production culture vaccine strain EV crop III I... generation: I generation - growing microbes in test tubes on a beveled PPP based on enzymatic hydrolysate of cattle meat (fwz), II generation - ZhPS-based fwz in vials III generation - cultivation in ZhPS-based fwz in bottles, the cultivation of native culture in ZhPS, vehicles - cultivators (reactors), the concentration of microbial suspension, the preparation of vaccine suspension using a protective environment drying of the following composition:

lactose, g·l -1

300

dextrin, g·l -1

30

thiourea, g·l -1

30

ascorbic acid, g·l -1

30

water distilled, l

until a definite volume,

lyophilized vaccine suspension at the following technological mode:

the temperature of the condenser - minus 50...70)C;

temperature of freezing material - minus 30 C;

depression in a drying - no more than 300 microns Hg;

drying speed - 2 C·h -1 ;

time dried -10 PM

In common with the claimed method is similar obtain the sowing of crops by the method of cultivation in ZhPS-based fwz in bottles and concentration of microbial suspension.

The main disadvantages of the prototype method is the following.

1. Obtaining traditional seed material, which includes a number of sequential transfers on solid and liquid nutrient mediums (crops I, II, III generations)that do not guarantee the stability of the basic biological properties and significantly reducing the resistance of vaccine strains of the plague microbe to the action of external factors at the subsequent stages of preparation of concentrated suspensions, vaccine suspension and the dry form of the finished product.

2. Application as a component of a protective environment drying dextrin, which is due to processes of coagulation and further sedimentation caused by high temperature in the autoclave, leads to an increase in optical density in comparison with the original. This further contributes to inadequate assessment of the actual concentration, increasing optical concentrations of microbial cells directly in vaccine preparation. The above facts lead to undesirable reduction one of the most important indicators of the quality of the vaccine plague live - percent live microbial cells.

3. The use of traditional mode of drying vaccine suspension with high death of microbes in the conditions of lyophilic stress.

All of the above disadvantages demanded serious search for optimal schemes of preparation of sowing material, ensuring stabilization of basic biological properties of microbes in the early stages of vaccine production, optimization of composition of the environment drying for the full protection of microbial cells prior to lyophilization and avoid possible technical errors in the determination of concentrations of microbial cells at the stage of preparation of vaccine suspension, testing regimes lyophilization, allowing to reduce the damaging effects of low temperature and dehydration on microbial cells in the drying process.

The task of the invention consists in reducing the duration of the process of obtaining vaccine drug with simultaneous improvement of its specific activity.

The problem is solved due to the fact that in the present method of receiving the vaccine is designed and optimized (table 1):

1. Scheme for seed culture strains of the plague microbe, involving the cultivation of fried stable starter cultures (PSC) in ZhPS in bottles.

For preparation PSC ampoule with reference crop strains of the plague microbe resuspendiruetsa to the original volume in distilled water, and then spend three consecutive testicular passages microbial culture through the organism is susceptible animals (Guinea pigs). Fried microbial culture sterile isolated from the organism of animals and mixed in a ratio of 2:1 with stabilizing liquid (glycerin, lactose and poliglyukin). Prepared PSK, designed to produce seed of culture in bottles bottled in bottles of 60...80 ml and stored in frozen state at a temperature of minus 18...22 C.

The cultivation of microbes in ZhPS in bottles should be performed within 48 hours at a temperature of 26...28 degrees C and continuous aeration at least 10 l·min-1 . After growing conduct biological control of culture and transmit it to the reactor for the preparation of native culture.

The comparative characteristic of native crops of vaccine strains of the plague microbe, prepared according to the claimed the scheme and scheme-the prototype presented in table 2.

2. The composition of a protective environment drying: lactose, g·l -1 - 300,0; thiourea, g·l -1 - 30,0; ascorbic acid, g·l -1 - 30,0; poliglyukin, g·l -1 - 30,0; distilled water, l - up to a definite volume, pH environment within...7,8 7,4% pH (table 3).

Poliglyukin (polysaccharide structure creators)-domestic dextran, obtained by the method of directed synthesis. Molecular weight poliglyukina is (50...70)·10 3 units Being a product of partial hydrolysis of dextrin, it has the same properties, but significantly lower molecular weight. Poliglyukin chemically inert, practically not involved in the connection with other substances. The safety, toxicity and apyrogenicity poliglyukina proved long and successful application of it in the field of gemotransfuziah. No side effects on human body allows its use as components of the environment drying for vaccines.

In the basis of its effect on microbial cells is the process of stabilization of the conformational state of protein molecules through the restoration of the intramolecular hydrogen bonds. Use poliglyukina as cryoprotectants can significantly reduce the destruction of microbial cells by freezing and dehydration in the process of freeze-drying.

3. Technological mode of freeze drying on the basic parameters: temperature of the condenser minus 60 C, temperature freezing material minus 40 C, vacuum drying chamber (depth vacuum) not more than 100 mm Hg, drying speed 3 C·h -1 , time of dried 6 h (table 3, figures 1-3).

The reduction in the duration of the process of obtaining vaccines, stabilization of the biological properties of the plague microbe on stages of preparation vaccine semi-finished products and improvement of qualitative characteristics of the dry form of the drug is due to:

1) the scheme of obtaining seed culture vaccine strain of the plague microbe, involving the cultivation PSC in ZhPS in bottles;

2) application-optimized component the composition of the protective environment drying;

3) optimized on the basic technological parameters regime lyophilization.

A causal relationship between populations of essential features of the claimed object and results presented in table 4.

The invention allows to prepare the vaccine corresponding to the requirements and stable biological properties throughout the valid storage period.

Possibility of realization of the claimed invention is shown by the following example.

For preparation of environment drying equipment poliglyukina pre pour 100...200 ml of warm 40...50 C distilled water and withstand 10...12 hours at a temperature of 18...24 C. Obtained homogeneous solution is filtered in a sterile vessel through gauze filter.

A portion of lactose pour 500 ml of distilled water and heated to 80...90 degrees Celsius with constant stirring until dissolved. After that the received solution is divided into two parts. In one part dissolve ascorbic acid, and the other with the thiourea; dissolution of thiourea is conducted at the temperature of 70 to 80 C. a Solution of ascorbic acid is neutralized 40% NaOH solution to the value of 7.3...7,5 pH units. Cooked solutions thiourea and ascorbic acid filtered through a gauze filter and is mixed with solution of poliglyukina. The pH mixture should be in the range of 7.2-7,6% pH. The adjustment of the pH-value produce 10% NaOH solution.

For adjustment of optical density in the preparation of vaccine suspension was prepared as additional solution environment drying with the content of components in 10 times less than the content in the main solution environment drying. Retrieved environment drying sterilized in an autoclave (fluid the ferry to 40 minutes; at 120 C to 20 min or filter on the filter equipment of the type "Sartorius" SM 16277 or Sartocon-mini").

In the bottle with a concentrated suspension of the plague microbe using vacuum add Wednesday drying the rate of 2:1 (2 parts of concentrated suspensions, 1 piece environment drying). The contents of the bottle thoroughly mixed shaking. After that, select a sample of vaccine suspension in the amount of 200...300 ml for assessing its biological properties. Vaccine suspension can be stored at a temperature of 2 to 6 degrees C, no more than 4 hours

Vaccine slurry is poured into a sterile penicillin vials, 2.0 ml each with automatic dosing over the flame of the spirit lamp.

Freeze-drying is carried out in the installation of TG-16. This 3 hours before download cool shelves camera to a temperature of minus 40 C, condenser to a temperature of minus 60 C. the Cell with bottles establish on 1-5 shelves sublimator. In one of the bottles on the shelves №1, 3, 5 insert the temperature sensor. Freezing of vaccine suspension spend up to a temperature of minus 40 C. After reaching the specified temperature vaccine suspension make the shutter speed for 2 hours Include vacuum pump and create negative pressure in the installation of not more than 100 mm Hg After 1 h after creating a specified value of depression include heated shelves. Then a gradual rise in temperature up to 0 C. the drying Rate in this period is 3 C·h -1 . When the temperature in the material 0 deg C with heating shelves for 6 h, bring it to a temperature of 25...30 C. The drying rate in this period is 5...6 degrees C·h -1 and final drying is carried out at this temperature for 6 hours

Comparative characteristics of experimental-production series of the vaccine plague living dry, established by the present method and the method prototype, and also in the process of long-term storage are presented in tables 5 and 6.

Table 2

The comparative characteristic of native cultures vaccine strains of the plague microbe (

X ± I 95 , n=5)

Scheme for the crop

The concentration of microbes, N·10 9 m CL·ml -1 , some...

The percentage of live microbial cells

Concentration of hydrogen ions pH units

on CCA turbidity gisk named after. L.A. Tarasevich

Cup method

Declare

24,0±2,0

12,1+1,3

50,4 7,7

Prototype

22,0+3,0

10,9±1,1

49,5 7,8 Table 3

Comparative characteristics of the stages of the technological process of obtaining plague vaccine

Name stages of the technological process

Description of the stages of the technological process

prototype (Industrial regulations IR # 2120-09)

the claimed object

TA - 14

The composition of the stabilizer:

The composition of the stabilizer:

Preparation of vaccine suspension

lactose, g·l -1 - 300;

lactose, g·l -1 - 300;

dextrin, g·l -1 - 30;

poliglyukin, g·l -1 - 30;

A BP-14-5

thiourea, g·l -1 - 30;

thiourea, g·l -1 - 30;

Preparation stabilizer

ascorbic acid, g·l -1 - 30;

ascorbic acid, g·l -1 - 30;

distilled water, l - up to a definite volume.

distilled water, l - up to a definite volume.

TP-15. Bottling vaccine suspension and sublimation drying

Technological mode of freeze drying on the basic parameters: temperature of the condenser minus 60 C;

Technological mode of freeze drying on the basic parameters: temperature of the condenser minus 60 C;

Operation TP-15-8-Sublimation drying

temperature of freezing material - minus 30 C; vacuum drying chamber (depth of vacuum) not more than 300 microns Hg; drying speed 2 degrees C·h -1 ; final 10 h

temperature of freezing material minus 40 C; vacuum drying chamber (depth vacuum) - not more than 100 mm Hg; drying speed 3 C·h -1 ; final time 6 hours

Table 4

A causal relationship between populations of essential features of the claimed object and achieved results

The technical result and their dimension

The actual and estimated

Detailed description, due to which it became possible improvement of the proposed facility in comparison with the prototype

prototype

the claimed object

The concentration of microbial cells,

Using optimized component composition protective environment drying and optimized the technological mode of freeze drying

billion·ml -1 : General (CCA turbidity GISC)

90 75

alive (Cup method)

24,5 26,1

The percentage of live microbial cells

27,2 34,8

Immunogenicity ED 50 , GML, for:

Use passivated stable starter cultures

white mice

11350 9053

Guinea pigs

7360 5225

The reduction in the duration of the process of obtaining vaccines, h

491 337

Use passivated stable starter cultures and optimized the technological mode of freeze drying

Table 5

Comparative characteristics of biological indicators vaccine plague living dry during long-term storage (

X ± I 95 , n=5)

The investigational vaccine series

The concentration of microbes, N·10 9 m CL·ml -1 , some...

The percentage of live microbial cells

Scheme for the crop

on CCA turbidity gisk named after. L.A. Tarasevich

Cup method

Freshly prepared

75,0±4,0

26,1±2,3

34,8 Declare

Stored for years...

1

75,0±3,0

25,7±1,9

34,2 2

75,0±2,0

25,4+2,6

33,8 3

75,0±2,0

23,3±2,0

31,1

Freshly prepared

90,0±2,0

24,5+1,3

27,2

Prototype

Stored for years...

1

90,0±2,0

23,8+1,5

26,4 2

90,0±2,0

23,2 ą1.5

25,7 3

90,0±2,0

22,5±1,8

25,0 Table 6

Characteristics of immunobiological properties of experimental-production series of the vaccine plague live dry

Quality score

Unit of measurement

The requirements of ND (Industrial regulations IR # 2120-09)

The results of the research series of the preparation, obtained using the method...

declare

prototype

The concentration of microbial cells per 1 ml:

on CCA turbidity risk

billion

From 50 to 100

75 90 live billion - 26,1 24,5

The percentage of live microbial cells

percentage

25,0, not less

34,8 27,2

The drug should

Specific safety

-

to be harmless with subcutaneous introduction of a Guinea pig mass (275±25)g 15 x 10 9 microbial cells (MCL) in 1 ml for CCA turbidity gisk named after. L.A. Tarasevich

The preparation is harmless with subcutaneous introduction of a Guinea pig mass (275±25) g 15 x 10 9 microbial cells (MCL) in 1 ml for CCA turbidity gisk named after. L.A. Tarasevich

Immunogenicity (ED 50 ):

40000, not more than

9053 11350

for white mice

GML

for Guinea pigs

10000, not more than

5225 7360

The way of reception of a preparation on the basis of the vaccine strain of the plague microbe, providing for the production of planting native culture of the plague microbe, the concentration of microbial suspension, cooking vaccine suspension and getting the dry form of the drug, wherein the preparation sowing of culture includes the cultivation of microbes in liquid nutrient medium in bottles within 48 hours at a temperature of 26...28C and continuous aeration at least 10 l·min -1 fried stable starter culture, the resulting three successive passages through the organism Guinea pigs and mixed in a ratio of 2:1 with stabilizing glycerine-lactose-polyglucinum fluid in the preparation of vaccine suspension use optimized component composition protective environment drying, including: lactose, g·l -1 - 300,0, thiourea, g·l -1 - 30,0, ascorbic acid, g·l -1 - 30,0; poliglyukin, g·l -1 - 30,0; distilled water, l - up to a definite volume, pH 7,4...7,8, and the lyophilization conduct the following mode: the temperature of the condenser minus 60 degrees C, the temperature of freezing minus material 40 C, the pressure in the drying chamber (depth vacuum) not more than 100 mm Hg, drying speed 3 C · h-1 and the time of final 6 o'clock

 

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