RU2123331C1 - Method of live antimeasles vaccine preparing - Google Patents

Abstract

FIELD: medicine, pharmacy, vaccines. SUBSTANCE: invention relates to production of antiviral vaccine preparations in tabletted form. Method involves measles virus multiplication on cellular substrate, collection of virus-containing fluid, cellular detritis removal and addition of stabilizing composition followed by lyophilization of a preparation. Virus-containing fluid is collected at virus titer 5.0-6.0 lg TCD (toxic cytopathic dose) 50/0.5 ml and at protein content 16 mcg/ml, not above. Sucrose, gelatose and peptone are added to virus-containing fluid as a stabilizing composition up to their final concentration in mixture by 3 wt.-% for each component. After lyophilization a dried virus-containing material is milled, mixed with lactose and calcium stearate at the following ratio of components, wt.-%: lyophilized virus-containing material at activity 4.5 lg TCD 50/0.5 ml, not less 49-98; lactose 0.1-49, and calcium stearate 1.5-2.5. Obtained mass is tabletted and tablets are covered with acid-resistant shell based on acetylphthalylcellulose. Prepared live antimeasles vaccine in tabletted form can be used for oral using. EFFECT: improved method of vaccine preparing, simple and economical equipment.

Description

 The invention relates to medicine, in particular, to the production of viral vaccine preparations in tablet form.

 A known method for producing live measles vaccine (LCV) based on any vaccine strain of measles virus. The method includes cultivating the virus in a culture of Japanese quail embryo cells, collecting virus-containing liquid, introducing a stabilizer and drying [1]. The vaccine induces the synthesis of hemagglutinating antibodies in humans and animals and provides a protective effect. The stabilizer contains sorbitol and gelatose.

The closest method (prototype) is the traditional technology for the production of LCS by propagating an attenuated measles virus strain L-16 strain on a Japanese quail fibroblast culture, followed by collection of the virus-containing liquid and its purification by filtration or centrifugation. A stabilizer is introduced into the resulting substance, including sorbitol and gelatose at a final concentration of 5% or LS-18 and gelatin. The product is poured into ampoules, freeze-dried, followed by sealing of the ampoules [2]. Before use, the vaccine is diluted with a solvent. The biological activity of the LCD is 3.3-4.0 lg TCD ₅₀ / 0.5 ml.

 The disadvantages of the known methods (analogue and prototype) for the production of ZhKV [1, 2] is the injection form of ZhKV, which poses a threat of infection with hepatitis viruses and HIV during vaccination, as well as the need for specially equipped rooms, a large number of syringes and needles, qualified personnel for vaccinations and revaccinations.

 The objective of the invention is to develop such a method for the production of LCS, which would make it possible to produce LCV in tablet form for oral use using simple and economical technological equipment.

This problem is solved by the fact that in the method for producing live measles vaccine, including the propagation of measles virus on a cellular substrate, the collection of virus-containing liquid, the release of the product from cellular detritus, the introduction of a stabilizing composition followed by lyophilization of the drug, according to the invention, the collection of virus-containing liquid is achieved a virus titer of at least 5.0-6.0 lg TCD ₅₀ / 0.5 ml and a protein content of not more than 16 μg / ml, sucrose, gelatose are introduced into the virus-containing liquid as a stabilizing composition and peptone to their final concentration in a mixture of 3 wt.% each stabilizer component, after lyophilization, the dry virus-containing material is crushed, mixed with lactose and calcium stearate in the following ratio of components, wt.%:
Lyophilized virus-containing material with an activity of at least 4.5 lg TCD ₅₀ / 0.5 ml - 49 - 98
Lactose - 0.1 - 49
Calcium Stearate - 1.5 - 2.5
The resulting mass is tabletted and the tablets are coated with an acid-resistant cellulose acetate-based coating.

 The vaccine prepared by the proposed method has pronounced immunogenic properties, tested experimentally in laboratory animals. During the production of the vaccine, the expensive and laborious stages of preparation and sterilization of ampoules, filling the vaccine into ampoules, their sealing, preparation and control of the solvent are excluded, as a result of which a number of production facilities and equipment are freed. A new form of vaccine release excludes the possibility of infection with hepatitis viruses and HIV during vaccination, the need for equipment for preventive vaccinations, provision of tools, and qualified personnel.

 The set of essential features of the invention, different from the essential features of the prototype, is unknown from published sources of information, which allows us to conclude that the technical solution meets the criterion of "inventive step".

 The method is illustrated by the following examples.

Example 1. The preparation of the tablet form of the LCD
The Japanese quail fibroblast cell culture is obtained by trypsinization of embryos with trypsin-versene solution at a temperature of + 37 ^o C. The cell suspension is centrifuged, the cells are resuspended in growth medium and scattered into mattresses or scooters at a rate of 500 and 950 thousand cells / ml, respectively. Cultivation is carried out at 35 ^o C to form a monolayer. In suspension infection, the virus is added to a suspension of cells with a multiplicity of infection of 0.1-0.001 TCD ₅₀ / cell. After the formation of a monolayer, the cells are subjected to 6-fold washing to release from ballast proteins, then they are cultured at a temperature of (35 ± 1) ^o C and a roller rotation speed of 2-12 rpm. When the cytopathic effect of the virus on cells appears, a virus-containing fluid is collected at intervals of 1-3 days, followed by replacement of the support medium. Individual viral plums with biological activity not lower than 5.0 lg TCD ₅₀ / 0.5 ml are combined and freed by filtration from cellular detritus. Sterile solutions of 50% sucrose, 25% gelatose (mol. Weight 3000) and 25% peptone are added to the resulting suspension to a final concentration of 3% of each compound. The composition is poured into trays of 1 l (liquid layer thickness 0.5 cm) and frozen at a temperature of -60 ^o C for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. The dried virus-containing material is crushed, mixed with lactose and calcium stearate at 49.0%, 49.0% and 2.0%, respectively. The resulting mass is tabletted and the tablets are coated with an acid-resistant cellulose acetate-based coating. The resulting vaccine is controlled by physico-chemical properties, the content of extraneous microflora, biological activity, harmlessness, antigenicity, immunogenicity (in laboratory animals).

Example 2. The results of the assessment of the physico-chemical properties of tablets of the LCD
The control of the tablet form of the LCD by the physicochemical properties is carried out in accordance with [2], the residual moisture is determined by the method of [3]. The average weight of the tablets is 0.2 - 0.5 g, the residual moisture content is (1 - 3)%. The tablets are strong, round, biconvex, without cracks, the edges are round without jagged places. Diameter 5 mm. The tablets do not disintegrate within 50 minutes in a 0.1N hydrochloric acid solution.

Example 3. Determination of the biological activity of the drug
To determine the biological activity, the virus is removed from the tablet into the aqueous phase (nutrient medium, phosphate-buffered saline, 8% aqueous sodium bicarbonate solution, followed by neutralization with 0.1N hydrochloric acid to pH 7.0-7.3), the aqueous phase is isolated centrifuged at 3000 rpm for 10 minutes and titrated in 96-well culture plates on a VERO cell culture according to the cytopathic effect in accordance with [2]. The biological activity of the tablet form of the LCD is 3.5 - 4.45 lg TCD ₅₀ / table.

Example 4. The determination of the content of extraneous microflora in the LCD
The methodology for analyzing the content of extraneous microflora in the LCD tablets is carried out in accordance with the Instruction [5].

 The content of foreign microorganisms is 1 to 5 colonies per tablet. No pathogenic bacteria of the intestinal group, Staphylococcus aureus, Pseudomonas aeruginosa were found.

Example 5. The study of the preparation of the LCD for toxicity
Toxicity in laboratory animals is determined in accordance with the procedure [6]. The tablets are ground in a sterile mortar with physiological saline (5 ml / table.), After settling for 15-30 minutes, the supernatant is injected subcutaneously in a dose of 5 ml to two outbred guinea pigs at the age of 3 months with a body weight of 300 ± 50 g and 0. 2 ml of five outbred white mice aged 2 months with a body weight of 17 - 20 g. All animals remained alive for 7 days, the weight of each animal did not decrease compared to the original on the day the experiment was completed, an abscess or necrosis did not develop at the injection site, no increase in temperature bottom of the animal.

Example 6. The study of the immunogenicity of the drug in animals
Control of the immunogenicity of the vaccine is carried out on rabbits. The drug is administered to animals orally. Studies show the effect of the vaccine on the stimulation of animal humoral and cellular immunity (in reactions of passive agglutination, inhibition of hemagglutination, ELISA, peripheral blood lymphocyte blast transformation) on days 14-21 after vaccination.

Experimental studies show that the tablet form of ZhKV prepared on the basis of a dry virus-containing preparation (activity 4.5 lg of TCV ₅₀ / 0.5 ml) obtained by traditional technology [1, 2] (with a stabilizer sorbitol with gelatin or LS-18 s gelatin) has biological activity and 2,5 lg 1.0 TCD _50/1 tablet respectively, less than one-dose vaccination (2000 TCD _50). The proposed technology for the production of a tablet form of LCV using a new stabilizer composition ensures the preservation of the biological activity of the virus-containing preparation.

Sources of information
1. USSR author's certificate N 278964, C 12 K 5/00, publ. 1973, "A method of obtaining a live measles vaccine" (analogue).

 2. FS 42 - 179 BC - 88, "Vaccine measles culture live, dry" (prototype).

3. The State Pharmacopoeia of the USSR. - M., 1987 .-- 11th ed. 2, p. 156
4. The State Pharmacopoeia of the USSR. - M., 1987 .-- 11th ed. 2, p. 196 - 209.

 5. A collection of instructions on general methods for monitoring sterility, physico-chemical properties, pyrogenicity, the absence of contaminating agents and the toxicity of MIBP. Order M3 of the USSR N 31 of 01/13/1983.

Claims (1)

A method of producing a live measles vaccine, including the propagation of measles virus on a cellular substrate, collecting virus-containing liquid, releasing the product from cellular detritus, introducing a stabilizing composition into it, followed by lyophilization of the drug, characterized in that the collection of virus-containing liquid is carried out upon reaching a virus titer of at least 5 - 6 lg TPD 50 / 0.5 ml and a protein content of not more than 16 μg / ml; sucrose, gelatin and peptone are introduced into the virus-containing liquid as a stabilizing composition to their final concentration in s hast to 3 wt% of each stabilizer component after lyophilization dry virus-containing material was pulverized, mixed with lactose and calcium stearate in the following ratio, wt.%.:
Lyophilized virus-containing material with an activity of at least 4.5 lg TPD 50 / 0.5 ml - 49 - 98
Lactose - 0.1 - 49
Calcium Stearate - 1.5 - 2.5
followed by tableting the resulting mass and coating with an acid-resistant coating based on acetylphthalyl cellulose.