CN102755642B - A kind of preparation method of anthrax vaccine - Google Patents

Abstract

The invention discloses a preparing method of live anthrax vaccines for people with percutaneous scarification, comprising the steps of: scraping a culture into water for injection when harvesting; blowing and/or shaking by using a suction tube to form a uniform suspension; adding equivoluminal sterile glycerol to the suspension; and uniformly mixing to obtain a stock solution.

Description

A kind of preparation method of anthrax vaccine

technical field

The invention relates to a preparation method of anthrax vaccine, in particular to a preparation method of live anthrax vaccine for human with skin scratches.

Background technique

Anthrax is a zoonotic acute infectious disease caused by Bacillus anthracis. It mainly exists in herbivores (sheep, cattle, horses, mules, donkeys, camels, etc.) and livestock communities, and causes extensive environmental pollution. Due to the strong resistance of Bacillus anthracis spores to the outside world, the pollution can continue to exist. Most people are infected by contact with sick animals or slaughtering and eating anthrax sick animals. Anthrax is prevalent all over the world, mainly in developing countries. Some countries have used Bacillus anthracis as a biological weapon. After 9/11, terrorists also used anthrax bacteria to carry out terrorist operations, which brought great panic to people.

The human anthrax vaccine used in my country is a live anthrax vaccine for humans with scratches on the skin. It is a live bacterial suspension prepared by culturing and collecting the attenuated strain of Bacillus anthracis and adding a stabilizer (50% glycerin solution) to dilute it. , the strain used is an attenuated strain of Bacillus anthracis CMCC63001 (A16R) which is non-capsulated, edematous, and has certain residual virulence. The third volume ^of the 2010 edition of the Pharmacopoeia ^of the People's Republic of China discloses the preparation method of the live anthrax vaccine for humans with scratches on the skin. The standard for the number of bacteria is not less than 16×10 ⁸ per 1 mL of live bacteria. However, the live bacteria rate of the vaccine prepared by this method is relatively low, especially in industrial production, the number of viable bacteria below 16×10 ⁸ /mL often occurs, the qualified rate of the product is low, and unqualified vaccines can only be discarded processing, which increases production costs.

Contents of the invention

One of the purposes of the present invention is to provide a preparation method of anthrax vaccine, especially the preparation method of live anthrax vaccine for people with scratches on the skin, which can easily obtain anthrax with high viable count and high viable rate live vaccines.

The inventors of the present invention have creatively proposed a new preparation method of live anthrax vaccine for humans with scratches on the skin through a large number of experimental studies. This method solves the technical problem of low viable count and viable bacterial rate in the existing preparation method.

Specifically, the present invention relates to a preparation method of anthrax live vaccine for humans with scratches on the skin, comprising the steps of:

When harvested, the culture is scraped into water for injection, pipetted and/or shaken to obtain a homogeneous suspension; and

Add an equal volume of sterile glycerin to the suspension, and mix well to obtain a stock solution.

Generally speaking, any shaking method known in the art or a combination thereof may be used for shaking, including but not limited to shaker shaking and/or manual shaking.

According to the method of the present invention, can comprise following steps before harvesting:

Inoculate a suitable medium with CMCC63001 (A16R) anthrax strains, and cultivate at 33-34°C for 18-24 hours, preferably 18-20 hours;

washing the culture, inoculating a suitable culture solution, and culturing at 33-34°C for 18-24 hours to obtain a seed culture; and

The seed culture is inoculated with a suitable medium, and cultured at 33-34°C until the mature typical spore formation rate reaches over 80%.

In addition, according to the method of the present invention, the following steps may also be included before harvesting:

Inoculate a suitable medium with CMCC63001 (A16R) anthrax strains, and cultivate at 33-34°C for 18-24 hours, preferably 18-20 hours;

Rinse the culture, inoculate a suitable medium, and incubate at 33-34°C for 18-24 hours;

washing the culture, inoculating a suitable culture solution, and culturing at 33-34°C for 18-24 hours to obtain a seed culture; and

The seed culture is inoculated with a suitable medium, and cultured at 33-34°C until the mature typical spore formation rate reaches over 80%.

Generally speaking, the culture time after the seed culture is inoculated with a suitable medium is generally 4-8 days, preferably 5-7 days.

In the method of the present invention, a suitable medium can be selected from thick Ginger agar medium, beef digestate agar medium, LB agar medium, brain heart infusion agar medium and nutrient agar medium, preferably thick gold Geer's agar medium and beef digestate agar medium; suitable culture medium can be selected from thick Ginger broth, beef digestate, LB culture fluid, brain heart infusion and nutrient broth, preferably thick Ginger meat Soup and beef digesta.

According to the method of the present invention, comprise following steps after harvesting:

Dilute the stock solution with 50% sterile glycerin solution to contain 40×10 ⁸ bacteria per 1 mL to obtain a semi-finished product; and

Finished products in batches and packaging.

According to the live anthrax vaccine prepared by the method of the present invention, the number of viable bacteria and the rate of viable bacteria are significantly higher than the existing method, for example, the number of viable bacteria is significantly higher than 16 × 10 ⁸ /mL, generally 30 × 10 ⁸ /mL Above, it can even reach above 38×10 ⁸ /mL, and its viable bacteria rate is obviously higher than 50%, generally above 80%, even above 95%. Therefore, the method of the invention greatly improves the qualified rate of products and reduces production costs.

Another advantage of the present invention is that it is easy to obtain a homogeneous suspension containing Bacillus anthracis at the time of harvest. Compared with the method in the prior art, which uses 50% glycerin solution to harvest, it needs shaker vibration or manual shaking for at least 2 hours to obtain a uniform suspension. The method of the present invention uses water for injection to harvest, which obviously saves manpower, material resources and Time costs.

Detailed ways

The following examples are only used to illustrate the present invention and cannot be used to limit the scope of the present invention.

Example 1 Comparison of using 50% glycerol solution and water for injection to harvest cultures to prepare live anthrax vaccines (shaker shaker and mix well)

1. Take 2 tubes of CMCC63001 (A16R) anthrax strains to inoculate thick Ginger agar medium, and incubate at 33-34°C for 18-24 hours.

2. Rinse the bacterial lawn with Thick Ginger Broth, inoculate in large tubes of Thick Ginger Broth, inoculate 2mL bacterial solution in each tube, and incubate at 33-34°C for 18-24 hours.

3. Collect the seed culture in the large tube of thick Ginger broth, inoculate it into thick Ginger agar Kjeldahl flasks, inoculate 40 of them in total, and inoculate 2mL bacterial solution in each Kjerkrower flask, 33-34 Cultivate at ℃.

4. Harvesting and preparing finished products

On the fifth day of thick Ginger agar Kirschner bottle culture, a bottle was randomly selected, and the bacterial lawn smear was taken for inspection. The mature typical spore formation rate was 93%. The cultures were harvested with 50% glycerol solution and water for injection to prepare anthrax live bacteria. vaccine.

4.1 Harvesting the culture using 50% glycerol solution to prepare live anthrax vaccine

4.1.1 Randomly take 20 Kelvin flasks to harvest, scrape the bacterial lawn into a flask filled with 60mL 50% sterile glycerin solution, add sterilized steel balls, shake at 4°C at 180 rpm, shake for 2 hours, and form a uniform suspension. Sampling for pure bacteria inspection (for the specific operation of pure bacteria inspection, refer to item 6.4 "Pure bacteria inspection" below, the same below), and then store the bacteria solution statically at 4°C.

4.1.2 Filter the samples that pass the pure bacteria test (14 days) with sterile gauze to obtain 50 mL of stock solution.

4.1.3 Preparation of semi-finished products: measure the concentration of the original solution to 68×10 ⁸ /mL (for the specific operation of determining the concentration, refer to the following item 5.2 "concentration determination method", the same below), add 34.6mL of 50% sterile Glycerin solution, mixed evenly, is a semi-finished product (prepared concentration is about 40×10 ⁸ /mL), which is checked for pure bacteria, and then repacked after passing the test.

4.1.4 Subpackage: Subpackage the semi-finished product into ampoules, 0.35mL/ampule, store at 4°C, batch number 20120101.

4.2 Production of Live Anthrax Vaccine by Harvesting Cultures Using Water for Injection

4.2.1 Harvest the remaining 20 Kelvin flasks, scrape the bacterial lawn into a flask filled with 40mL water for injection, add sterilized steel balls, shake on a shaker at 4°C at 180 rpm, shake for 2 hours to form a uniform suspension, and take samples Perform a pure bacteria test, and then store the bacteria solution at 4°C.

4.2.2 Filter the samples that pass the pure bacteria test (14 days) with sterile gauze to obtain a uniform suspension of 33 mL, add an equal volume of sterile glycerin and mix to obtain a stock solution.

4.2.3 Preparation of semi-finished products: measure the concentration of stock solution to be 54×10 ⁸ /mL, add 22.9mL of 50% sterile glycerin solution to 65.5mL of stock solution, mix well to obtain semi-finished products (preparation concentration is about 40×10 ⁸ /mL), carry out Check for pure bacteria, and repack after passing the test.

4.2.4 Subpackage: Subpackage the semi-finished product into ampoules, 0.35mL/ampule, store at 4°C, batch number 20120102.

5. Determination of the number of viable bacteria, concentration and calculation of viable bacteria rate

5.1 Determination of the number of viable bacteria

5.1.1 Take 3 vaccines, combine them and mix them evenly.

5.1.2 Take 0.5mL of the above combined solution, add 3.5mL of water for injection to obtain an 8-fold diluted bacterial solution, and then dilute the 8-fold diluted bacterial solution as follows:

Take 0.5mL of 8-fold diluted bacterial solution + 4.5mL of water for injection (10 ^-1 bacterial solution);

Take 0.5mL of 10 ^-1 bacteria solution + 4.5mL of water for injection (10 ^-2 bacteria solution);

Take 0.5mL of 10 ^-2 bacteria solution + 4.5mL of water for injection (10 ^-3 bacteria solution);

Take 0.5mL of 10 ^-3 bacteria solution + 4.5mL of water for injection (10 ^-4 bacteria solution);

Take 0.5mL of 10 ^-4 bacteria solution + 4.5mL of water for injection (10 ^-5 bacteria solution);

Take 0.5mL of 10 ^-5 bacterial solution + 4.5mL of water for injection (10 ^-6 bacterial solution).

5.1.3 Inoculation: Inoculate 0.5mL of ^10-6 bacterial solution on 5 thick Ginger agar medium plates, 0.1mL per plate, spread evenly with an "L" stick immediately after inoculation, and incubate at 35-37°C Cultivate in the box for 24 hours, and one colony represents one viable bacterium.

5.1.4 Calculation of results:

The number of viable bacteria (per 1mL) = the sum of the number of viable bacteria in 5 plates × 2 × 10 ⁶ × 8.

5.2 Concentration determination method

5.2.1 Take 3 vaccines, combine them and mix them evenly.

5.2.2 Take 0.5 mL of the above combined solution and add 4.5 mL of 1% formalin saline to obtain a 10-fold diluted bacterial solution, and incubate at 37°C for 2 hours.

5.2.3 Take 0.5 mL of the 10-fold diluted bacterial solution and add 3.5 mL of normal saline to obtain an 80-fold diluted bacterial solution.

5.2.4 Use the reference product for the determination of anthrax vaccine concentration distributed by the National Drug Control Agency to measure the concentration of 80-fold diluted bacterial solution by spectrophotometry with a wavelength of 600nm.

5.2.5 Calculation of results:

The concentration of the vaccine=the concentration of the 80-fold diluted bacterial solution×80.

5.3 Calculation of live bacteria rate

The calculation formula of viable bacteria rate is: viable bacteria rate = number of viable bacteria/concentration of vaccine.

See Table 1 for the number of live bacteria, the results of the concentration determination and the calculation results of the live bacteria rate of the vaccines with batch numbers 20120101 and 20120102 determined according to the above method.

Table 1 Comparison of viable bacteria count, concentration and viable bacteria rate

Note: The concentration standard is: 3.2×10 ⁹ to 4.8×10 ⁹ bacteria per 1 mL, that is, 32×10 ⁸ to 48×10 ⁸ ;

The standard for the number of viable bacteria is: the number of viable bacteria per human dose (50 μL) should not be less than 0.8×10 ⁸ , that is, not less than 16×10 ⁸ /mL.

6. Quality inspection

6.1 Stability investigation

After storing the two batches of vaccines for half a year, measure the number and concentration of viable bacteria, and calculate the rate of viable bacteria. The method is the same as the above item 5. The results are shown in Table 2.

Table 2 The number of live bacteria, concentration and live bacteria rate comparison

6.2 Identification test

Check with the plate method, inoculate the two batches of vaccines on the thick Ginger agar medium, add 1 drop of anthrax phage at the working concentration, and observe that there is no anthracis bacillus growth in the two batches of vaccines where the phages were dropped.

6.3 Appearance

After observation, it was found that the two batches of vaccines were all gray-white homogeneous suspensions, and did not contain any bacteria lumps or foreign matter that could not disperse.

6.4 Pure bacteria inspection

Two batches of vaccines were inoculated on nutrient agar, thioglycollate and modified Martin's medium respectively, each of which was cultured at 20-25°C and 30-35°C for 14 days, and the growth was examined by smear microscope, and 2 The growths of the batches of vaccines all conformed to the characteristics of the Bacillus anthracis CMCC63001 (A16R) strain and were free of miscellaneous bacteria.

6.5 Specific Toxicity Test

Two vaccines were taken from each of the two batches, combined and prepared into a suspension with a concentration of 2.5×10 ⁸ /mL, and stored at 4°C for future use.

Take 10 rabbits with a body weight of 2.0-2.5 kg and divide them into 2 groups randomly, with 5 rabbits in each group. Each rabbit in the first group is subcutaneously injected with 1 mL of the suspension prepared by the vaccine with the batch number 20120101, and each rabbit in the other group is injected subcutaneously with 1 mL of the vaccine with the batch number 1mL of the suspension prepared for the 20120102 vaccine. After 10 days of observation, it was found that all the experimental animals survived, and some animals had edema at the injection site.

Conclusion: The live anthrax vaccine prepared by harvesting the culture with water for injection has significantly higher number and rate of live bacteria than the vaccine prepared by harvesting the culture with 50% glycerol solution. There was no significant difference in the quality of the vaccines prepared by the two methods in terms of stability inspection, identification test, appearance, pure bacteria inspection and specific toxicity inspection.

Example 2 Comparison of using 50% glycerol solution and water for injection to harvest cultures to prepare live anthrax vaccines respectively (shaking and mixing by hand)

1. Take two CMCC63001 (A16R) anthrax strains and inoculate thick Ginger agar medium, and incubate at 33-34°C for 18-20 hours.

2. Rinse the bacterial lawn with Thick Ginger Broth, inoculate in large tubes of Thick Ginger Broth, inoculate 2mL bacterial solution in each tube, and incubate at 33-34°C for 18-24 hours.

3. Collect the seed culture in the large tube of thick Ginger broth, inoculate it into thick Ginger agar Kjeldahl flasks, inoculate 30 of them in total, and inoculate 2m L of bacterial solution in each Kjerkrower flask, 33-34 Cultivate at ℃.

4. Harvesting and preparing finished products

4.1 Harvesting: On the fifth day of thick Ginger agar Kirschner bottle culture, randomly select a bottle, take a smear of the bacterial lawn for inspection, the mature typical spore formation rate is 92%, use 50% glycerol solution and water for injection to harvest the culture respectively Preparation of live anthrax vaccine.

4.1.1 Randomly take 15 Kelvin flasks to harvest, scrape the bacterial lawn into a flask filled with 45mL 50% sterile glycerin solution, add sterilized steel balls, and name it as sample A;

4.1.2 Harvest the remaining 15 Kelvin flasks, scrape the bacterial lawn into a flask filled with 30mL water for injection, add sterilized steel balls, and name it as sample B;

4.1.3 Another experimenter manually shakes the bacterial liquids harvested in 4.1.1 and 4.1.2 for 10 minutes in a blinded state (without knowing the difference between the two bacterial liquids), and samples are taken for pure bacteria inspection, and then The bacterial solution was stored at 4°C for 14 days, during which time it was manually shaken for 10 minutes every day to obtain a uniform suspension.

4.2 Filter sample A that passed the pure bacteria test (14 days) with sterile gauze to obtain 35 mL of stock solution; filter sample B with sterile gauze to obtain 23 mL of uniform suspension, add an equal volume of sterile glycerin and mix to obtain stock solution.

4.3 Preparation of semi-finished products

4.3.1 Preparation of Sample A

Measure the concentration of the original solution to be 73×10 ⁸ /mL, add 28.5mL of 50% sterile glycerin solution to 34.5mL of the original solution, mix well to obtain a semi-finished product (prepared concentration is about 40×10 ⁸ /mL), conduct a pure bacteria test, and pass the test Subpackage.

4.3.2 Preparation of Sample B

The concentration of the stock solution was determined to be 56×10 ⁸ /mL, and 18.2mL of 50% sterile glycerin solution was added to 45.5mL of the bacterial solution, and the semi-finished product was obtained after mixing evenly (the prepared concentration was about 40×10 ⁸ /mL). Subpackage later.

4.4 Subpackage: Subpackage the semi-finished product into ampoules, 0.35mL/ampule, and store at 4°C. The batch numbers of sample A and sample B are 20120403 and 20120404, respectively.

5. Determination of the number of viable bacteria, concentration and calculation of viable bacteria rate

Carry out viable count and concentration determination with reference to the method for the 5th item of embodiment 1, and calculate viable rate, the results are shown in Table 3.

Table 3 Viable bacteria count, concentration and viable bacteria rate comparison

Note: The standard for the number of viable bacteria is: the number of viable bacteria per human dose (50 μL) should not be less than 0.8×10 ⁸ , that is, not less than 16×10 ⁸ /mL.

Conclusion: The live anthrax vaccine prepared by harvesting the culture with water for injection by manual shaking and mixing has significantly higher number and rate of viable bacteria than the vaccine prepared by harvesting the culture with 50% glycerol solution and shaking and mixing by hand. In addition, the number of viable bacteria in this batch of vaccine prepared by harvesting the culture using 50% glycerol solution did not meet the quality standard.

Example 3 Preparation of Live Anthrax Vaccine by Harvesting Cultures Using Water for Injection (Pipe and mix with a pipette and shake by hand)

1. Take 2 tubes of CMCC63001 (A16R) anthrax strains and inoculate beef digestate agar medium, and incubate at 33-34°C for 18-24 hours.

2. Rinse the bacterial lawn with beef digestive juice, inoculate in large tubes of beef digestive juice, inoculate 2mL bacterial solution in each tube, and incubate at 33-34°C for 18-24 hours.

3. Collect the seed culture in the large tube of beef digestion solution, inoculate it into beef digestion solution agar Kirschner flasks, inoculate 30 in total, inoculate 2mL bacterial solution in each flask, and incubate at 33-34°C.

4. Harvesting and preparing finished products

4.1 Harvesting: On the fifth day of culturing beef digestate agar Kirschner flasks, randomly select a bottle and take a smear of the bacterial lawn for inspection. The mature typical spore formation rate is 95%. The 30 Kirschner flasks are randomly divided into two groups for harvesting. Scrape the bacterial lawn into a flask filled with 30mL of water for injection. One is named sample A, and it is blown with a straw for 1 minute to mix to form a homogeneous suspension; the other is named sample B, and sterilized steel balls are added, and shaken manually for 10 minutes to mix. into a uniform suspension, samples were taken for pure bacteria inspection, and then stored at 4°C.

4.2 Filter samples A and B that passed the pure bacteria test (14 days) with sterile gauze to obtain a uniform suspension of 24 mL, respectively, add an equal volume of sterile glycerin and mix to obtain a stock solution.

4.3 Preparation of semi-finished products

4.3.1 Preparation of Sample A

Measure the concentration of the original solution to be 58×10 ⁸ /mL, add 21.4mL of 50% sterile glycerin solution to 47.5mL of the original solution, and mix well to obtain a semi-finished product (prepared concentration is about 40×10 ⁸ /mL), conduct a pure bacteria test, and pass Subpackage later.

4.3.2 Preparation of Sample B

Measure the concentration of the original solution to be 57×10 ⁸ /mL, add 20.2mL of 50% sterile glycerin solution to 47.5mL of the original solution, mix well to obtain a semi-finished product (prepared concentration is about 40×10 ⁸ /mL), conduct a pure bacteria test, and pass the test Subpackage.

4.4 Subpackage: Subpackage the semi-finished product into ampoules, 0.35mL/ampule, and store at 4°C. The batch numbers of sample A and sample B are 20120505 and 20120506, respectively.

5. Determination of the number of viable bacteria, concentration and calculation of viable bacteria rate

Carry out viable count and concentration determination with reference to the method for the 5th item of embodiment 1, and calculate viable rate, the results are shown in the following table 4.

Table 4 The number of live bacteria, concentration and live bacteria rate comparison

Conclusion: The live anthrax vaccine prepared by harvesting the culture with water for injection can obtain a vaccine with a relatively high number of viable bacteria and a relatively high rate of viable bacteria just by pipetting for 1 minute or manually shaking for 10 minutes. This shows that compared with the method of using 50% glycerol solution to harvest cultures to prepare live anthrax vaccines, this method can obviously save manpower, material resources and time costs.

Example 4 Preparation of Live Anthrax Vaccine by Harvesting Cultures at Different Time Points Using Water for Injection

1. Take 2 tubes of CMCC63001 (A16R) anthrax strains to inoculate thick Ginger agar medium, and incubate at 33-34°C for 18-24 hours.

2. Rinse the bacterial lawn with Thick Ginger Broth, inoculate in large tubes of Thick Ginger Broth, inoculate 2mL bacterial solution in each tube, and incubate at 33-34°C for 18-24 hours.

3. Collect the seed culture in the large tube of thick Ginger broth, inoculate it into thick Ginger agar Kjeldahl flasks, inoculate 30 of them in total, and inoculate 2m L of bacterial solution in each Kjerkrower flask, 33-34 Cultivate at ℃.

4. Harvesting and preparing finished products

4.1 Harvest on the fifth day

4.1.1 On the fifth day of thick Ginger agar Kirschner flask culture, randomly select a bottle and take a smear of the bacterial lawn for inspection. The mature typical spore formation rate is 93%. Randomly pick 15 Kirschner flasks and harvest the bacterial lawn. Scrape into a flask filled with 30mL of water for injection, blow it with a straw for 1 minute to mix, take a sample for pure bacteria inspection, and then store the bacterial solution at 4°C for 14 days, during which time, shake manually for 1 minute every day to obtain a uniform suspension.

4.1.2 Filter the samples that pass the pure bacteria test (14 days) with sterile gauze to obtain a uniform suspension of 25 mL, add an equal volume of sterile glycerin and mix to obtain a stock solution.

4.1.3 Preparation of semi-finished products: measure the concentration of the stock solution to be 56×10 ⁸ /mL, add 19.8mL of 50% sterile glycerin solution to 49.5mL of the stock solution, mix well to obtain a semi-finished product (preparation concentration is about 40×10 ⁸ /mL), carry out Check for pure bacteria, and repack after passing the test.

4.1.4 Subpackage: Subpackage the semi-finished product into ampoules, 0.35mL/ampule, store at 4°C, batch number is 20120607.

4.2 Harvest on the seventh day

4.2.1 On the seventh day of thick Ginger agar Kirschner flask culture, randomly select a bottle, and take a smear of the bacterial lawn for inspection. The mature typical spore formation rate is 95%. Take the remaining 15 Kirschner flasks to harvest, and the bacterial lawn Scrape into a flask filled with 30mL of water for injection, blow with a straw for 1 minute to mix, take a sample for pure bacteria inspection, and then store the bacterial solution at 4°C for 14 days, during which time, shake manually for 1 minute every day to obtain a uniform suspension.

4.2.2 Filter the samples that pass the pure bacteria test (14 days) with sterile gauze to obtain a uniform suspension of 24 mL, add an equal volume of sterile glycerin and mix to obtain a stock solution.

4.2.3 Preparation of semi-finished products: measure the concentration of the stock solution to be 55×10 ⁸ /mL, add 17.8mL of 50% sterile glycerin solution to 47.5mL of the stock solution, mix well to obtain the semi-finished product (preparation concentration is about 40×10 ⁸ /mL), carry out Check for pure bacteria, and repack after passing the test.

4.2.4 Subpackage: Subpackage the semi-finished product into ampoules, 0.35mL/ampule, store at 4°C, the batch number is 20120608.

5. Determination of the number of viable bacteria, concentration and calculation of viable bacteria rate

Carry out viable count and concentration determination with reference to the method for the 5th item of embodiment 1, and calculate viable rate, the results are shown in the following table 5.

Table 5 The number of live bacteria, concentration and live bacteria rate comparison

Conclusion: The live anthrax vaccine prepared by harvesting the culture with water for injection has a higher number of viable bacteria and a higher rate of viable bacteria no matter whether it is cultured for five days or seven days.

Embodiment 5 respectively uses physiological saline and water for injection harvesting culture to prepare the comparison of anthrax live vaccine

1. Take 2 tubes of CMCC63001 (A16R) anthrax strains to inoculate thick Ginger agar medium, and incubate at 33-34°C for 18-24 hours.

2. Rinse the bacterial lawn with Thick Ginger Broth, inoculate in large tubes of Thick Ginger Broth, inoculate 2mL bacterial solution in each tube, and incubate at 33-34°C for 18-24 hours.

3. Collect the seed culture in the large tube of thick Ginger broth, inoculate it into thick Ginger agar Kjeldahl flasks, inoculate 30 of them in total, inoculate 2mL of bacterial liquid in each Kjeldahl flask, and inoculate at 33-34°C nourish.

4. Harvesting and preparing finished products

4.1 Harvesting: On the fifth day of thick Ginger agar Kirschner flask culture, a bottle was randomly selected, and the bacterial lawn smear was taken for inspection. The mature typical spore formation rate was 94%. The 30 Kirschner flasks were randomly divided into two groups for harvesting, of which One group scraped the bacterial lawn into a flask containing 30 mL of normal saline, named it Sample A, and mixed it with a straw for 1 minute to form a uniform suspension; the other group scraped the bacterial lawn into a flask containing 30 mL of water for injection, Name it sample B, blow it with a pipette for 1 minute to mix it into a uniform suspension, take samples for pure bacteria inspection, and then store it at 4°C.

4.2 Filter samples A and B that pass the pure bacteria test (14 days) with sterile gauze to obtain a uniform suspension of 23 mL, respectively, add an equal volume of sterile glycerin and mix to obtain a stock solution.

4.3 Preparation of semi-finished products

4.3.1 Preparation of Sample A

The concentration of the original solution was determined to be 57×10 ⁸ /mL, and 19.3mL of 50% sterile glycerin solution was added to 45.5mL of the original solution, and the semi-finished product was obtained after mixing evenly (the prepared concentration was about 40×10 ⁸ /mL). Subpackage later.

4.3.2 Preparation of Sample B

Measure the concentration of the original solution to be 56×10 ⁸ /mL, add 18.2mL of 50% sterile glycerin solution to 45.5mL of the original solution, mix well to obtain a semi-finished product (prepared concentration is about 40×10 ⁸ /mL), conduct a pure bacteria test, and pass the test Subpackage.

4.4 Subpackage: subpackage the semi-finished product into ampoules, 0.35mL/ampule, store at 4°C, the batch numbers of sample A and sample B are 20120609 and 20120610 respectively.

5. Determination of the number of viable bacteria, concentration and calculation of viable bacteria rate

Carry out viable count and concentration determination with reference to the method for the 5th item of embodiment 1, and calculate viable rate, the results are shown in the following table 6.

Table 6 Viable bacteria count, concentration and viable bacteria rate comparison

Conclusion: The live anthrax vaccine prepared by harvesting the culture with water for injection has significantly higher number and rate of live bacteria than the vaccine prepared by harvesting the culture with normal saline, and the live vaccine prepared by harvesting the culture with normal saline The number does not meet quality standards.

In the above-mentioned examples, unless otherwise specified, all operations can refer to the Pharmacopoeia of the People's Republic of China (2010 edition).

Claims (23)

1. a kind of preparation method of scratch human anthrax live vaccine on the skin, comprises the steps: When harvested, the culture is scraped into water for injection, pipetted and/or shaken to obtain a homogeneous suspension; and Add an equal volume of sterile glycerin to the suspension, and mix well to obtain a stock solution. 2. The method according to claim 1, wherein, comprising the steps before harvesting: Inoculate a suitable medium with CMCC63001 (A16R) anthrax strains and incubate at 33-34°C for 18-24 hours; washing the culture, inoculating a suitable culture solution, and culturing at 33-34°C for 18-24 hours to obtain a seed culture; and The seed culture is inoculated with a suitable medium, and cultured at 33-34°C until the mature typical spore formation rate reaches more than 80%. 3. The method of claim 1, wherein, before harvesting, comprise the steps of: Inoculate a suitable medium with CMCC63001 (A16R) anthrax strains and incubate at 33-34°C for 18-24 hours; Rinse the culture, inoculate a suitable medium, and incubate at 33-34°C for 18-24 hours; washing the culture, inoculating a suitable culture solution, and culturing at 33-34°C for 18-24 hours to obtain a seed culture; and The seed culture is inoculated with a suitable medium, and cultured at 33-34°C until the mature typical spore formation rate reaches more than 80%. 4. The method of claim 1, wherein, before harvesting, comprise the steps of: Inoculate a suitable medium with CMCC63001 (A16R) anthrax strains and incubate at 33-34°C for 18-20 hours; washing the culture, inoculating a suitable culture solution, and culturing at 33-34°C for 18-24 hours to obtain a seed culture; and The seed culture is inoculated with a suitable medium, and cultured at 33-34°C until the mature typical spore formation rate reaches more than 80%. 5. The method of claim 1, wherein, before harvesting, comprise the steps of: Inoculate a suitable medium with CMCC63001 (A16R) anthrax strains and incubate at 33-34°C for 18-20 hours; Rinse the culture, inoculate a suitable medium, and incubate at 33-34°C for 18-24 hours; washing the culture, inoculating a suitable culture solution, and culturing at 33-34°C for 18-24 hours to obtain a seed culture; and The seed culture is inoculated with a suitable medium, and cultured at 33-34°C until the mature typical spore formation rate reaches more than 80%. 6. The method according to any one of claims 2 to 5, wherein the suitable medium is selected from thick Ginger agar medium, beef digestate agar medium, LB agar medium, brain heart infusion agar culture Medium and nutrient agar medium; suitable culture medium is selected from Thick Ginger Broth, Beef Digest, LB Broth, Brain Heart Infusion and Nutrient Broth. 7. The method as claimed in claim 6, wherein, suitable substratum is selected from thick Ginger agar medium and beef digestate agar medium; suitable nutrient solution is selected from thick Ginger broth and beef digestate . 8. The method according to any one of claims 1 to 5, wherein the shaking is carried out by means of shaker oscillation and/or manual shaking. 9. The method according to claim 6, wherein the shaking is carried out by shaker shaking and/or manual shaking. 10. The method according to any one of claims 1 to 5, wherein, after harvesting, comprising the steps of: Dilute the stock solution with 50% sterile glycerin solution to contain 40×10 ⁸ bacteria per 1 mL to obtain a semi-finished product; and Finished products in batches and packaging. 11. The method of claim 6, wherein, after harvesting, comprising the steps of: Dilute the stock solution with 50% sterile glycerin solution to contain 40×10 ⁸ bacteria per 1 mL to obtain a semi-finished product; and Finished products in batches and packaging. 12. The method of claim 8, wherein, after harvesting, comprising the steps of: Dilute the stock solution with 50% sterile glycerin solution to contain 40×10 ⁸ bacteria per 1 mL to obtain a semi-finished product; and Finished products in batches and packaging. 13. The method of claim 9, wherein, after harvesting, comprising the steps of: Dilute the stock solution with 50% sterile glycerin solution to contain 40×10 ⁸ bacteria per 1 mL to obtain a semi-finished product; and Finished products in batches and packaging. 14. The method according to any one of claims 2-5, wherein the culture time after the seed culture is inoculated with a suitable medium is 4-8 days. 15. The method as claimed in claim 14, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 5-7 days. 16. The method as claimed in claim 6, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 4-8 days. 17. The method according to claim 16, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 5-7 days. 18. The method according to claim 8, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 4-8 days. 19. The method according to claim 18, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 5-7 days. 20. The method according to claim 9, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 4-8 days. 21. The method according to claim 20, wherein the culture time after the seed culture is inoculated with a suitable medium is 5-7 days. 22. The method according to claim 13, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 4-8 days. 23. The method according to claim 22, wherein the cultivation time after the seed culture is inoculated with a suitable medium is 5-7 days.