RU2259214C1 - Method for preparing inactivated dry smallpox vaccine "ospavir" - Google Patents

Abstract

FIELD: biotechnology, vaccines.

SUBSTANCE: method involves preparing industrial strain, smallpox scrape off and virus-containing liquid. Virus-containing liquid is inactivated by gamma-radiation Co⁶⁰. Integral (total) dose of irradiation is 1.75 x 10⁶ R at the parent specific activity of virus-containing liquid from 1 x 10⁹ to 1.9 x 10⁹ OOE/ml and 1.9 x 10⁶ R at the parent specific activity of virus-containing liquid from 2 x10⁹ to 5 x 10 OOE/ml. The content of total protein is brought about to the concentration 100 mcg/ml and drying stabilizing agents are added. Vaccine elicits high immunogenicity, suitable in applying and absence of toxicity.

EFFECT: improved preparing method, improved and valuable properties of vaccine.

3 cl, 6 ex

Description

The invention relates to microbiology and can be used to obtain vaccine smallpox inactivated dry.

A known method of producing vaccine smallpox live, including obtaining industrial strains, smallpox scrapings, virus-containing fluids (production schedule of smallpox live vaccine dry No. 963-00 from 09/14/2000). Also known is a method of producing a vaccine of smallpox inactivated (production schedule for inactivated vaccine No. 249 of 1982).

However, the use of smallpox live vaccine is limited by a number of established contraindications, especially in conditions where the population has not been vaccinated for a long time or has not previously been vaccinated against smallpox.

The objective of the proposed method is the creation of grossimmunity in a person vaccinated for the first time against smallpox, and reducing the reactogenicity of the smallpox vaccine alive.

The problem is achieved by obtaining a production strain, smallpox scrapings, virus-containing liquid and its inactivation, moreover, the vaccine virus is inactivated by gamma radiation of Co ⁶⁰ , the integral (total) dose of radiation is 1.75 · 10 ⁶ R at the initial specific activity of the virus-containing liquid 1 · 10 ⁹ -1.9 · 10 ⁹ OOE / ml or 1.9 · 100 ⁶ P - at the initial specific activity of the virus-containing liquid 2 · 10 ⁹ -5 · 10 ⁹ OOE / ml, the total protein content is adjusted to a concentration of 100 μg / ml, drying stabilizers are introduced into the composition, as abilizatorov drying sucrose is used at a final concentration of 5% and zhelatozu a final concentration of 1%, is used as production strain of vaccinia virus strain A-TTI (Lister Institute of Viral Preparations).

The vaccine process includes:

- obtaining production strains;

- receiving smallpox scrapings;

- preparation of virus-containing fluid;

- preparation of inactivated viral fluid;

- preparation of a liquid semi-finished vaccine - mixing inactivated viral fluid, buffer solution, stabilizer solutions;

- pouring liquid semi-finished product into ampoules, lyophilization of the vaccine and sealing the ampoules with the vaccine;

- packaging of the vaccine complete with a solvent, which is used as a solution of sodium chloride 0.9%.

Obtaining production strains is:

- obtaining a production strain by passage of the original strain of the vaccine virus in rabbits, obtaining scraping, grinding, treatment with Freon-113, obtaining a virus-containing liquid with the addition of glycerin in a final concentration of 50% (lapine);

- obtaining a uterine strain by passage of a production strain (lapins) on calves, obtaining a scraping, grinding it, processing with Freon-113, obtaining a virus-containing liquid with the addition of glycerin in a final concentration of 50% (lapino-vaccine of the 1st generation);

- receiving the inoculum virus by passage on the calves of the uterine strain, obtaining scraping, grinding, treatment with Freon-113, obtaining virus-containing fluid with the addition of glycerol in a final concentration of 50% (lapino-vaccine of the II generation).

The specific activity of production strains is controlled in the reaction on the chorion-allantoic membranes of twelve-day-old chicken embryos and should be: lapins — not less than 5.0 · 10 ⁸ OOE / ml, lapino-vaccines I and II generation — not less than 1.0 · 10 ⁹ OOE / ml

Obtaining smallpox scrapings is the production of smallpox detritus during passage of the seed virus in calves.

Preparation of virus-containing fluid:

- grinding the scraping, processing the scraping with Freon-113 homogenization at 5-10 thousand rpm for 10 minutes, obtaining a virus-containing fluid (lyapino-vaccine III generation) in the form of a supernatant after centrifugation at 3000 rpm for 20 minutes.

The specific activity of the generation III lapino vaccine is controlled in the reaction on the chorion-allantoic membranes of twelve-day-old chicken embryos and should be at least 1.0 · 10 ⁹ OOE / ml. Microbiological purity is controlled in accordance with the Global Fund XI issue 2: no more than 50 extraneous microorganisms are allowed in 1 ml, including aerobic and facultative anaobor bacteria and fungi. There should be no pathogenic anaerobic bacteria, bacteria of the Enterobacteriaceae family and Pseudomonas aeruginosa and Staphylococcus aureus species. The material should be harmless to guinea pig and two white mice.

The preparation of inactivated viral fluid is the process of inactivating a virus with gamma radiation. Virus-containing liquid is irradiated in an isotopic installation using Co ⁶⁰ .

The integral (total) dose of exposure to the virus-containing liquid does not depend on its volume and amounts to 1.75 · 10 ⁶ R at the initial specific activity of the virus-containing liquid 1 · 10 ⁹ OOE / ml - 1.9 · 10 ⁹ OOE / ml or 1.9 · 10 ⁶ P at the initial specific activity of the virus-containing liquid 2 · 10 ⁹ OOE / ml - 5 · 10 ⁹ OOE / ml

Inactivated viral fluid should not contain live virus, should be sterile, non-toxic and contain a total protein of at least 1 mg / ml.

Monitoring the completeness of inactivation is carried out on the chorion-allantoic membranes of twelve-day-old chicken embryos, on which typical specific lesions from the application of inactivated viral fluid should not develop.

Preparation of liquid prefabricated vaccine:

A series of liquid prefabricated vaccines is obtained by adding stabilizers to an inactivated viral liquid with a calculated total protein concentration of 100 μg / ml.

Stabilizers: gelatose at a final concentration of 1% and sucrose at a final concentration of 5% are administered in the form of solutions of gelatose 10% and sucrose 50%.

The concentration of total protein in inactivated viral fluid to 100 μg / ml is adjusted with Mc Ilvein's buffer solution of 0.007 M pH 7.2-7.4.

Spilling liquid prefabricated into ampoules, freeze-drying the vaccine and sealing the ampoules with the vaccine:

The drug is poured into sterile ampoules. The nominal volume of the vaccine in the ampoule during its spill is 0.5 ml. The spill process is carried out in a stream of sterile air.

Then the drug cassettes are placed in a low-temperature counter to freeze the product to a final temperature of minus 50-55 ° C. The frozen preparation is lyophilized for 36 hours. The initial temperature of the shelves is minus 40 ° С, the heating rate of the shelves is 5 ° С, the final product temperature is 25 ° С.

The dried preparation is sealed in a stream of sterile nitrogen to protect against oxygen in the air. The concentration of residual oxygen in the preparation after sealing should be no more than 3%.

Vaccine packaging complete with solvent:

The vaccine is supplied complete with a solvent - 0.9% sodium chloride solution. Primary vaccine packaging - 1 dose (0.5 ml) in ampoules ШП-1, ШП-2 made of glass grade НС-1 or НС-3 or of similar quality, or imported, approved for use in the Russian Federation, solvent packaging - 0 , 5 ml in the same ampoules.

Vaccines with a vaccine and solvent are labeled with fast-curing ink by printing or labeling.

The kit consists of one vaccine ampoule and one solvent ampoule. For 5 or 10 sets, together with the instructions for use and an ampoule knife or a scarifier, they are packed in a pack of cardboard boxed or in blister packs. One or two contour packs are placed in a pack of cardboard.

The main quality indicator of a vaccine characterizing immunobiological properties is specific activity. The specific activity of the drug, due to the active component, the inactivated antigen of the vaccine virus, is evaluated by the ability to induce the formation of virus-neutralizing antibodies to the vaccine virus in immunized white rats. The concentration of specific virus-neutralizing antibodies (titer) in the blood serum of immunized animals is determined in the biological neutralization reaction on chicken embryos using the vaccine virus as a test strain. The geometric mean titer of virus-neutralizing antibodies in the blood serum of rats vaccinated with a single dose of the vaccine intravenously should be at least 1:80.

As a production strain for producing inactivated smallpox vaccine, the L-IVP vaccine virus strain is used.

We offer examples of the preparation of a series of liquid prefabricated vaccines.

, составляет

c или 21 час 40 минут.Example 1. Virus-containing liquid obtained after processing scraping (RP No. 963-00 from 09/14/00) has a specific activity of 1.0 · 10 ⁹ OOE / ml The virus is inactivated in an isotope installation with Co ⁶⁰ gamma radiation at an integrated radiation dose (D) of 1.75 · 10 ⁶ R. At a radiation dose rate (W) of 22.45 R / s, the inactivation duration (T) calculated by the formula

is

c or 21 hours 40 minutes.

Inactivated viral fluid should be safe (control the completeness of inactivation), sterile, non-toxic and the total protein content should be at least 100 μg / ml. In an example of a concentration of total protein, 3.0 mg / ml was established. To prepare a series of semi-finished vaccines with a volume of 15.55 L, it is necessary to prepare 12.5 L of inactivated viral fluid with a calculated total protein concentration of 100 μg / ml. To do this, 417 ml of inactivated viral fluid with a protein concentration of 3.0 mg / ml is adjusted to 12500 ml with McIlvein's sterile pyrogen-free non-toxic buffer solution of 0.007 M pH 7.2-7.4 (estimated amount of added buffer solution is 12083 ml). The liquid is stirred to prevent foaming, and a 20 ml recess is taken to control the concentration of total protein. Then stabilizers are added to the obtained liquid: gelatose to a final concentration of 1% in the form of a sterile pyrogen-free non-toxic solution of gelatose 10% - 1560 ml and sucrose to a final concentration of 5% in the form of a sterile pyrogen-free non-toxic solution of sucrose 50% - 1560 ml. The resulting liquid is mixed and a 50 ml sample is taken for sterility control. A series of liquid prefabricated vaccines with a volume of 15.55 L is transferred for filling into ampoules.

составляет

с или 20 часов 15 минут.Example 2. Virus-containing liquid obtained after processing scraping (RP No. 963-00 from 09/14/00) has a specific activity of 1.5 · 10 ⁹ OOE / ml The virus is inactivated in an isotope installation with Co ⁶⁰ gamma radiation at an integrated radiation dose (D) of 1.75 · 10 ⁶ R. At a radiation dose rate (W) of 24 R / s, the inactivation duration (T) calculated by the formula

makes up

from or 20 hours 15 minutes.

Inactivated viral fluid should be safe (control the completeness of inactivation), sterile, non-toxic, and the total protein content should be at least 100 μg / ml. In the example, the concentration of total protein is set to 2.0 mg / ml. To prepare a series of semi-finished vaccines with a volume of 15.55 L, it is necessary to prepare 12.5 L of inactivated viral fluid with a calculated total protein concentration of 100 μg / ml. For this, 625 ml of inactivated viral fluid with a protein concentration of 2.0 mg / ml is adjusted to 12500 ml with McIlvein's sterile pyrogen-free non-toxic buffer solution of 0.007 M pH 7.2-7.4 (estimated amount of buffer solution added is 11875 ml). The liquid is stirred to prevent foaming, and a 20 ml recess is taken to control the concentration of total protein. Then stabilizers are added to the obtained liquid: gelatose to a final concentration of 1% in the form of a sterile pyrogen-free non-toxic solution of gelatose 10% - 1560 ml and sucrose to a final concentration of 5% in the form of a sterile pyrogen-free non-toxic solution of sucrose 50% - 1560 ml. The resulting liquid is mixed and a 50 ml sample is taken for sterility control. A series of liquid prefabricated vaccines with a volume of 15.55 L is transferred for filling into ampoules.

, составляет

или 18 часов 41 минуту.Example 3. Virus-containing fluid obtained after processing scraping (RP No. 963-00 from 09/14/00) has a specific activity of 1.9 · 10 ⁹ OOE / ml The virus is inactivated in an isotope installation with Co ⁶⁰ gamma radiation at an integrated radiation dose (D) of 1.75 · 10 ⁶ R. At a radiation dose rate (W) of 26 R / s, the inactivation duration (T) calculated by the formula

is

or 18 hours 41 minutes.

Inactivated viral fluid should be safe (control the completeness of inactivation), sterile, non-toxic and the total protein content should be at least 100 μg / ml. In the example, the concentration of total protein is set to 3.25 mg / ml. To prepare a series of semi-finished vaccines with a volume of 15.55 L, it is necessary to prepare 12.5 L of inactivated viral fluid with a calculated total protein concentration of 100 μg / ml. For this, 385 ml of inactivated viral fluid with a protein concentration of 3.25 mg / ml is adjusted to 12500 ml with McIlvein's sterile pyrogen-free non-toxic buffer solution of 0.007 M pH 7.2-7.4 (estimated amount of buffer solution added is 12115 ml). The liquid is stirred to prevent foaming, and a 20 ml recess is taken to control the concentration of total protein. Then stabilizers are added to the obtained liquid: gelatose to a final concentration of 1% in the form of a sterile pyrogen-free non-toxic solution of gelatose 10% - 1560 ml and sucrose to a final concentration of 5% in the form of a sterile pyrogen-free non-toxic solution of sucrose 50% - 1560 ml. The resulting liquid is mixed and a 50 ml sample is taken for sterility control. A series of liquid prefabricated vaccines with a volume of 15.55 L is transferred for filling into ampoules.

, составляет

или 22 часа.Example 4. Virus-containing liquid obtained after processing scraping (RP No. 963-00 from 09/14/00) has a specific activity of 2.0 · 10 ⁹ OOE / ml The virus is inactivated in an isotope installation with Co ⁶⁰ gamma radiation at an integrated radiation dose (D) of 1.9 · 10 ⁶ R. At a radiation dose rate (W) of 24 R / s, the inactivation duration (T) calculated by the formula

is

or 22 hours.

Inactivated viral fluid should be safe (control the completeness of inactivation), sterile, non-toxic and the total protein content should be at least 100 μg / ml. In an example of a total protein concentration, 3.5 mg / ml was established. To prepare a series of semi-finished vaccines with a volume of 15.55 L, it is necessary to prepare 12.5 L of inactivated viral fluid with a calculated total protein concentration of 100 μg / ml. For this, 357 ml of inactivated viral fluid with a protein concentration of 3.5 mg / ml is adjusted to 12500 ml with McIlvein's sterile pyrogen-free non-toxic buffer solution of 0.007 M pH 7.2-7.4 (the estimated amount of buffer solution added is 12143 ml). The liquid is stirred to prevent foaming, and a 20 ml recess is taken to control the concentration of total protein. Then stabilizers are added to the obtained liquid: gelatose to a final concentration of 1% in the form of a sterile pyrogen-free non-toxic solution of gelatose 10% - 1560 ml and sucrose to a final concentration of 5% in the form of a sterile pyrogen-free non-toxic solution of sucrose 50% - 1560 ml. The resulting liquid is mixed and a 50 ml sample is taken for sterility control. A series of liquid prefabricated vaccines with a volume of 15.55 L is transferred for filling into ampoules.

, составляет

или 23 часа 28 минут.Example 5. The virus-containing fluid obtained after processing the scraping (RP No. 963-00 from 09/14/00) has a specific activity of 4.4 · 10 ⁹ OOE / ml The virus is inactivated in an isotope installation with Co ⁶⁰ gamma radiation at an integrated radiation dose (D) of 1.9 · 10 ⁶ R. At a radiation dose rate (W) of 22.45 R / s, the inactivation duration (T) calculated by the formula

is

or 23 hours 28 minutes.

Inactivated viral fluid should be safe (control the completeness of inactivation), sterile, non-toxic and the total protein content should be at least 100 μg / ml. In an example of a concentration of total protein, 3.125 mg / ml was established. To prepare a series of semi-finished vaccines with a volume of 15.55 L, it is necessary to prepare 12.5 L of inactivated viral fluid with a calculated total protein concentration of 100 μg / ml, for which 400 ml of inactivated viral fluid with a protein concentration of 3.125 mg / ml is adjusted to 12500 ml with sterile pyrogen-free non-toxic McIlvein buffer solution 0.007 M pH 7.2-7, A (estimated amount of buffer solution added is 12100 ml). The liquid is stirred to prevent foaming, and a 20 ml recess is taken to control the concentration of total protein. Then stabilizers are added to the obtained liquid: gelatose to a final concentration of 1% in the form of a sterile pyrogen-free non-toxic solution of gelatose 10% - 1560 ml and sucrose to a final concentration of 5% in the form of a sterile pyrogen-free non-toxic solution of sucrose 50% - 1560 ml. The resulting liquid is mixed and a 50 ml sample is taken for sterility control. A series of liquid prefabricated vaccines with a volume of 15.55 L is transferred for filling into ampoules.

, составляет

или 20 часов 17 минут.Example 6. Virus-containing fluid obtained after processing scraping (RP No. 963-00 from 09/14/00) has a specific activity of 5.0 · 10 ⁹ OOE / ml The virus is inactivated in an isotope installation with Co ⁶⁰ gamma radiation at an integrated radiation dose (D) of 1.9 · 10 ⁶ R. At a radiation dose rate (W) of 26 R / s, the inactivation duration (T) calculated by the formula

is

or 20 hours 17 minutes.

Inactivated viral fluid should be safe (control the completeness of inactivation), sterile, non-toxic and the total protein content should be at least 100 μg / ml. In the example, the concentration of total protein is set to 2.5 mg / ml. To prepare a series of semi-finished vaccines with a volume of 15.55 L, it is necessary to prepare 12.5 L of inactivated viral fluid with a calculated total protein concentration of 100 μg / ml. For this, 500 ml of inactivated viral fluid with a protein concentration of 2.5 mg / ml is adjusted to 12500 ml with McIlvein's sterile pyrogen-free non-toxic buffer solution of 0.007 M pH 7.2-7.4 (estimated amount of added buffer solution is 12000 ml). The liquid is stirred to prevent foaming, and a 20 ml recess is taken to control the concentration of total protein. Then stabilizers are added to the obtained liquid: gelatose to a final concentration of 1% in the form of a sterile pyrogen-free non-toxic solution of gelatose 10% - 1560 ml and sucrose to a final concentration of 5% in the form of a sterile pyrogen-free non-toxic solution of sucrose 50% - 1560 ml. The resulting liquid is mixed and a 50 ml sample is taken for sterility control. A series of liquid prefabricated vaccines with a volume of 15.55 L is transferred for filling into ampoules.

The vaccine does not contain a preservative. The vaccine is dried under vacuum from a frozen state in ampoules and sealed under nitrogen. Obtained by the proposed method, the vaccine "Ospavir" can be used in medicine for vaccination against smallpox.

The advantages of the resulting vaccine are that the vaccine has a single dose package in an ampoule. Purpose: primary vaccination with a two-stage vaccination method for the active prevention of smallpox in children from two years of age, adolescents and adults. The vaccine is given to persons subject to primary vaccination. In addition, the Ospavir vaccine has an advantage over a similar multidose vaccine due to its simplicity and ease of use, as well as the elimination of iatrogenic error.

Claims (3)

1. A method of obtaining a vaccine of smallpox inactivated dry by obtaining a production strain, smallpox scrapings, virus-containing liquid and its inactivation, characterized in that the virus-containing liquid is inactivated by Co ⁶⁰ gamma radiation, wherein the integral radiation dose is 1.75 · 10 ⁶ P at the initial the specific activity of the virus-containing liquid 1 · 10 ⁹ -1.9 · 10 ⁹ OOE / ml and 1.9 · 10 ⁶ P - with the initial specific activity of the virus-containing liquid from 2 · 10 ⁹ to 5 · 10 ⁹ OOE / ml, then the content is adjusted total protein to concentration 100 μg / ml and drying stabilizers are added. 2. The method according to claim 1, characterized in that sucrose in a final concentration of 5% and gelatose in a final concentration of 1% are used as drying stabilizers. 3. The method according to claim 1, characterized in that the strain of the vaccine virus L-IVP is used as the production strain.