CN116077634A - Dispersing and freeze-drying method for bacillus calmette-guerin thallus for treatment, stock solution, semi-finished product and finished product - Google Patents

Dispersing and freeze-drying method for bacillus calmette-guerin thallus for treatment, stock solution, semi-finished product and finished product Download PDF

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CN116077634A
CN116077634A CN202211538816.0A CN202211538816A CN116077634A CN 116077634 A CN116077634 A CN 116077634A CN 202211538816 A CN202211538816 A CN 202211538816A CN 116077634 A CN116077634 A CN 116077634A
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treatment
bcg
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drying
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江秋虹
张健
张保钱
王维为
朱战
徐守腾
梁永培
陶立峰
王国治
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Anhui Zhifei Longcom Biopharmaceutical Co ltd
Chongqing Zhifei Biological Products Co Ltd
Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
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Anhui Zhifei Longcom Biopharmaceutical Co ltd
Chongqing Zhifei Biological Products Co Ltd
Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
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Abstract

The invention belongs to the field of vaccine preparation, and particularly discloses a method for dispersing and freeze-drying bacillus calmette-guerin bacteria for treatment, stock solution, semi-finished product and finished product, wherein 15 g-20 g of bacteria are dispersed into 125 g-167 g of freeze-drying protective agent to obtain the stock solution of bacillus calmette-guerin for treatment, the concentration of bacterial suspension with 1 unit weight is 120 mg/mL-140 mg/mL; dispersing the stock solution of the BCG vaccine for treatment by 1.8-2.5 times by adopting a freeze-drying protective agent, and uniformly mixing to obtain a BCG vaccine semi-finished product for treatment with 1 unit weight; the concentration of the BCG bacterium in the 1 unit weight of the semi-finished product of the BCG vaccine for treatment is 48-72 mg/mL. The quality of the BCG vaccine freeze-dried product for treatment prepared by the invention is far higher than the standard required by three parts of Chinese pharmacopoeia, and experiments prove that the live bacteria number of the BCG vaccine for treatment related by the invention is also greatly improved, the BCG vaccine freeze-dried product for treatment has higher initial live bacteria number and thermal stability, and therefore, the perfusion dosage is possibly reduced, and the shortage problem of the product is relieved.

Description

Dispersing and freeze-drying method for bacillus calmette-guerin thallus for treatment, stock solution, semi-finished product and finished product
Technical Field
The invention belongs to the field of vaccine preparation, and in particular relates to a method for dispersing and freeze-drying bacillus calmette-guerin bacteria for treatment, and a stock solution, a semi-finished product and a finished product thereof.
Background
The BCG vaccine for treatment is a preparation prepared by culturing BCG bacteria to collect thalli, preparing high-concentration BCG bacterial suspension, adding a stabilizer and freeze-drying. Currently, 5 products are marketed worldwide, and major suppliers of northland and europe announce formally exiting the market in 2017 due to regulatory, cost and equipment factors, resulting in a shortage of therapeutic BCG worldwide. The stability of the current freeze-dried product can be kept for 18 months at the highest, the actual market demand can not be met in 18 months of effective period in practical application, and the shelf life of the product is short, so that the production of the BCG vaccine for treatment with high quality and good stability is a challenging work.
The BCG used for the prior market of the Bisaiji treatment is prepared by culturing BCG used for collecting thalli, adding a stabilizer, subpackaging into an ampoule, and freeze-drying to prepare an immunotherapeutic agent, wherein the lyoprotectant is sucrose, gelatin, sodium glutamate and potassium chloride, the same freeze-drying production process as that of BCG vaccine for preventing tuberculosis (the current edition of Chinese pharmacopoeia) is accepted in the industry, the viable count of BCG bacteria and the stability of the viable count, which are one of the quality indexes of BCG vaccine products for treatment, are key factors influencing the immune effect, each bottle contains 60mg of BCG bacteria, and each 1mg of BCG bacteria contains 1.0x10 viable count of BCG bacteria 6 The quality of CFU and viable count can only meet the requirements of the current edition three of the Chinese pharmacopoeia of not less than 1.0X10 6 CFU/mg "minimum standard; and the ampoule is adopted to interior package material, on the one hand because of stability when package material is upright is poor, and the semi-manufactured goods partial shipment back business turn over freeze dryer can't accomplish automatic business turn over when freeze-drying can't adopt manual business turn over freeze dryer, does not accord with automated production requirement, brings the risk for product safety, and on the other hand has the gap with the mainstream package material xilin bottle that this kind of product adopted internationally.
Currently, in the research on the bacillus calmette guerin in the prior art, the research on the prescription of a protective agent and the research on the freeze-drying process in the research on the freezing and freeze-drying technology of the bacillus calmette guerin are mostly aimed at. For example, patent publication No. CN106754376A proposes a microbial low-temperature preservation protective agent, and a preparation method and application thereof; wherein the components of the microbial low-temperature preservation protective agent are as follows: reducing sugar, glycerol, nonionic surfactant, potassium salt, glutamate and water; meanwhile, a technical method for low-temperature long-term preservation of the bacillus calmette-guerin is established, and a technical support is provided for deep development of related researches of the bacillus calmette-guerin; however, the BCG vaccine for treatment basically adopts freeze-dried injection, has poor liquid freezing stability and can not meet the clinical use requirement. The patent with publication number CN1218750C discloses a protective agent for treating BCG vaccine and a freeze-drying process, and the prepared combined vaccine has strong immunogenicity; publishing a recombinant hIFN-alpha-2 b-BCG lyoprotectant prescription: 10% of sucrose, 1% of gelatin, 1% of sodium glutamate and 1% of potassium chloride, the freeze-dried preparation has high viable bacteria rate, meets the biological immune treatment standard, and has biological characteristics and stability of plasmids. Patent with publication number of CN111588859A proposes a lyoprotectant and application thereof, a lyoprotectant seedling and a preparation method thereof, wherein the lyoprotectant comprises the following components in percentage by weight: 10% of sucrose, 0.5-2.0% of dextran, 1% of sodium glutamate and 1% of KCL, or 10% of sucrose, 1% of dextran, 1-5% of trehalose, 1% of sodium glutamate and 1% of KCL, wherein the solvent for preparing the freeze-drying protective agent is water for injection, and the pH value of the freeze-drying protective agent is regulated to 7.3-7.5 by 10% of sodium hydroxide solution. However, the stability research result of the treatment prepared by the method of the patent shows that the effective quality time is 3 months, which is far lower than the longest period of 18 months of the commercial products. After the thalli are dispersed by adopting a protective agent, the concentration of the semi-finished thalli is 120mg/mL. In summary, it is clear that the study of the dispersion pattern of the bacterial cells in the protective agent is not involved.
Existing bcg vaccine freeze-drying processes generally include three steps of freezing, first-stage sublimation drying (primary drying) and second-stage analytical drying (secondary drying), and studies indicate that freezing and primary drying are one of the most critical stages in the freeze-drying process. The freeze-drying process does not consider that the BCG vaccine for treatment is a high-concentration freeze-dried live bacteria preparation, the content of solid substances (the sum of the content of active ingredients and the content of non-active ingredients) in the prescription of the preparation is 19% (g/g), and the components and proportion of the freeze-drying protective agent in the semi-finished product, the dispersion mode of thalli in the protective agent and the freeze-drying process have important influences on the content of live bacteria after freeze-drying of the product and the stability of heat resistance and storage resistance. In addition, the freeze-drying process adopts a conventional rapid cooling method, in the method, the temperature difference between the bottom layer and the surface layer of the same bottle product in contact with the laminate is larger, the bottom layer reaches the eutectic point of the product, the surface layer is far away, and crystals are formed from the bottom to the surface layer instantaneouslyAnd then re-dissolving and re-crystallizing. Thus, the solution concentration of different layers of the same bottle product is different, a high-concentration cover is formed on the surface layer, the sublimation of the product after pre-freezing is not facilitated, the phenomena of poor physical properties, formation of larger crystals, bright surface layer, bottom atrophy and the like are easily caused, and the mechanical damage to the frozen thalli can be caused by repeated freezing and thawing. Thus leading to large freeze-drying loss of the product, and the measurement result of viable count shows that the viable count is not less than 1.0X10 6 CFU/mg, but the value reaches a critical value, there is a risk of failure. Therefore, gradient cooling is usually adopted in the freezing stage, for example, a freeze-drying method of a live vaccine is proposed in the patent with publication number of CN107543373A, annealing treatment is introduced between the freezing and the drying steps, the obtained product has no large ice crystals, good physical properties and titers of the product are easy to sublimate, the non-uniformity and the drying rate between the same bottle and different bottles are improved, the glass transition temperature of the frozen concentrate of the amorphous item is improved, and the stability of the product is improved. A primary drying stage, during which most of the water is sublimated; the analysis and drying stage is to remove residual moisture, and if the bacillus calmette guerin is not well controlled, the appearance, the moisture, the quality and the stability of the living bacteria are directly affected. Conventional control methods at relevant stages are disclosed in the acknowledged lyophilization process of BCG vaccine for treatment and the patent with publication number of CN111588859A, but the influence on the viable count and heat-resistant stability of the product is not considered, so that the viable count or stability is influenced.
In summary, the concentration of the semi-finished product of the BCG vaccine for treatment is 120mg/mL, and any control method for thallus dispersion in the preparation process of the stock solution and the semi-finished product is not disclosed, but the invention needs to find a method for preparing the semi-finished product through thallus dispersion, and needs to find a method for freeze-drying the BCG vaccine for treatment instead of the annealing step in the prior art, so that the defect of low quality or stability of the BCG vaccine for treatment is overcome, and the BCG vaccine for treatment can relieve the current shortage and has more industrialization scale and safety on the basis.
Disclosure of Invention
In view of the above problems, in a first aspect, the present invention provides a method for dispersing bacillus calmette-guerin bacteria for treatment, wherein 15 g-20 g of bacteria are dispersed in 125 g-167 g of freeze-drying protective agent to obtain a bacillus calmette-guerin stock solution for treatment with a 1 unit weight bacterial suspension concentration of 120 mg/mL-140 mg/mL;
dispersing the BCG vaccine stock solution for treatment by 1.8-2.5 times by adopting a freeze-drying protective agent, and uniformly mixing to obtain a BCG vaccine semi-finished product for treatment with 1 unit weight;
the concentration of the BCG bacterium in the 1 unit weight of the semi-finished product of the BCG vaccine for treatment is 48-72 mg/mL.
Further, the lyoprotectant comprises 10% of sucrose, 1% of gelatin, 1% of sodium glutamate, 1% of potassium chloride and the balance of water for injection.
Further, the method for obtaining the stock solution of the BCG vaccine for treatment comprises the following steps:
grinding 15 g-20 g BCG strain according to the mass ratio of the BCG strain to 1000g steel balls;
and (3) grinding, adding 125-167 g of freeze-drying protective agent, and uniformly mixing for 10 minutes to obtain the stock solution of the BCG vaccine for treatment.
Further, the grinding conditions were: the rotating speed is 180-200 r/min, and the time is 3-5 min.
Further, the mass ratio of the bacillus calmette-guerin thallus to the freeze-drying protective agent is 17-20 g:125 g-135 g.
Further, the mass ratio of the bacillus calmette-guerin thallus to the lyoprotectant is 19.8g:130g.
Further, the concentration of the BCG bacterium in the 1 unit weight of the semi-finished product of the BCG vaccine for treatment is 54-66 mg/mL.
Still further, the 1 unit weight of the therapeutic BCG semi-finished product has a BCG concentration of 60mg/mL.
In a second aspect, the invention provides a bcg stock solution for treatment, wherein the weight of the bcg stock solution for treatment is 1-120 times of the 1 unit weight of the bcg stock solution.
In a third aspect, the invention provides a therapeutic bcg vaccine semi-finished product, wherein the weight of the therapeutic bcg vaccine semi-finished product is 1-120 times of the unit weight of the therapeutic bcg vaccine semi-finished product.
In a third aspect, the invention provides a method for lyophilizing a semi-finished product of BCG vaccine for treatment,
the freeze-drying method is that the semi-finished product of the BCG vaccine for treatment enters a freeze-drying box under the precooling condition of less than or equal to minus 40 ℃, the heat preservation is not more than 2 hours after all the semi-finished products enter the box, annealing, sublimation drying and analysis drying are sequentially carried out, and the finished product of the BCG vaccine for treatment is obtained after the capping is carried out by pressing plug.
Further, the annealing is: setting the temperature of the partition plate to be any one of-10 to-14 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 1 to 6.5 hours; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 3-5 h, wherein the time for reaching the final temperature is not more than 0.5 h;
The sublimation drying is as follows: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be increased to-20 ℃, and the time for reaching the final temperature is 4-5 hours; continuously adjusting the temperature of the partition plate to-16 ℃ and keeping the temperature for 6-11 hours, wherein the time for reaching the final temperature is 2.5-3 hours; the vacuum degree is controlled to be 10-20 pa;
the analysis and drying stage is as follows: the temperature of the partition board is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 3-5 h; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 1-2.5 h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree in the process at 10-20 pa, and preserving the heat for 4-9 h, wherein the vacuum degree in the heat preservation process is not higher than 5pa.
Still further, the annealing is: setting the temperature of the partition board to be minus 14 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 1 to 6.5 hours; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 3-5 h, wherein the time for reaching the final temperature is not more than 0.5 h;
the sublimation drying is as follows: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 10-20 pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 4-5 h; continuously adjusting the temperature of the partition plate to-16 ℃ and keeping the temperature for 6-11 hours, wherein the time for reaching the final temperature is 2.5-3 hours; the vacuum degree is controlled to be 10-20 pa;
The analysis and drying stage is as follows: the temperature of the partition board is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching 0 ℃ is 3-5 h; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 1-2.5 h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree in the process at 10-20 pa, and preserving the heat for 4-9 h, wherein the vacuum degree in the heat preservation process is not higher than 5pa.
In a fifth aspect, the invention also provides a finished product of the BCG vaccine for treatment, which is prepared by adopting the freeze-drying method.
The invention has the beneficial effects that:
the quality of the BCG vaccine freeze-dried product for treatment prepared by the invention is far higher than the standard required by the three parts of Chinese pharmacopoeia, and experiments prove that the quality of the BCG vaccine viable count for treatment related by the invention is higher than the standard required by the three parts of Chinese pharmacopoeia by not less than 1.0x10 6 CFU/mg standard, live bacillus calmette-guerin number average for 5 batches of treatment of 7.36×10 6 CFU/mg; the heat stability test result shows that the quality of the viable bacteria rate is higher than the three requirements of Chinese pharmacopoeia and is not lower than 25 percent and not lower than 2.5X10 5 CFU/mg standard, live bacterial rate average value of BCG vaccine for 5 batches of treatment is 49.8%, live bacterial number average value is 6.86×10 6 CFU/mg has higher initial viable count and thermal stability, and thus it is possible to reduce the infusion dose, alleviating the shortage problem of such products.
The BCG vaccine freeze-dried product for treatment prepared by the invention has higher stability, and 5 batches of BCG vaccine for continuous treatment are placed for 24 months at the temperature of 5+/-3 ℃, and the average value of the number of viable bacteria in each batch is 6.24 multiplied by 10 6 CFU/mg, far above 1.0X10 6 CFU/mg, the viable bacteria rate of the thermal stability test is not lower than 25%; standing at 25deg.C+ -2deg.C for 6 months, wherein the number average of viable bacteria in each batch is 3.06X10 6 CFU/mg; standing at 37deg.C+ -2deg.C for 3 months, wherein the number average of viable bacteria remaining in each batch is 1.4X10 6 CFU/mg, all of which are not lower than 1.0X10 6 CFU/mg, thereby maintaining continuous stability of product quality during storage, transport and use in high temperature areas.
The BCG vaccine freeze-dried product for treatment prepared by the invention has higher effectiveness, and experiments prove that the bladder perfused female C57BL/6J mouse MB49 bladder cancer cells have tumor inhibiting effect, can obviously inhibit proliferation of the bladder cancer MB49 cells, obviously enhance cell immunity compared with a control dispersion method and freeze-dried technical products, and obviously promote the secretion of BCG-PPD specific IFN-gamma and IL-2 cell number of the mouse spleen lymphocytes.
The bacillus calmette-guerin vaccine freeze-dried product for treatment prepared by the invention adopts an inner wrapping material of a penicillin bottle, is connected with an international main stream wrapping material, has stronger international competitiveness, and is proved by experiments to be placed for 24 months at 5+/-3 ℃ and has the moisture content lower than 2.0 percent and higher than the quality standard which is not higher than 3.0 percent and is required by the three parts of Chinese pharmacopoeia.
The invention aims at the freeze-drying procedure of the BCG vaccine for treatment, adopts a specific freeze-drying curve, and ensures that the BCG vaccine for treatment is well freeze-dried and formed by parameter control in each stage of pre-freezing (annealing), sublimation drying and analytical drying, and the freeze-dried finished product meets the three requirements of Chinese pharmacopoeia.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention adopts a stabilizer and strain which are consistent with those of the existing market products, and provides a bacillus calmette-guerin thallus dispersing method for treatment, a freeze-drying process and application thereof.
The invention is further described below in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. In each of the examples described below, the reagents used in the examples were commercially available unless otherwise specified. BCGD 2 PB 302 S II The strain A10 is from Chinese food and drug inspection institute. The reagents such as sucrose, sodium glutamate, KCL, gelatin and the like are all of pharmaceutical grade.
EXAMPLE 1 cell culture
(1) And (3) strain recovery:
opening BCGD in biological safety cabinet 2 PB 302 S II Adding 1-5 mL of 0.9% sodium chloride injection into 10 working seeds of the first strain to prepare bacterial suspension, uniformly mixing, absorbing bacterial liquid, inoculating the bacterial suspension onto the inclined plane of a Rogowski egg culture medium, inoculating 0.2mL of each Rogowski egg culture medium, and standing and culturing for 21-23 days at 37-39 ℃. The growth state of the cells was observed, and samples were taken for cell detection (acid-fast staining).
Wherein, 1035ml of the Luo's egg culture medium is prepared by the following steps:
400mlL-J matrix formulation usage amount: 0.15g of magnesium sulfate, 7.5ml of glycerol, 4.5g of sodium glutamate, 1.5g of monopotassium phosphate, 18.8g of potato starch, 0.375g of magnesium citrate and 375ml of water;
the L-J matrix is uniformly mixed, then is water-bath and is shaken while being heated until the L-J matrix is transparent pasty. The L-J matrix and 2% malachite green solution are respectively sterilized by high-pressure steam at 121 ℃ for 30min, 625ml of fresh egg liquid is added into the L-J matrix, evenly mixed, 10ml of 2% malachite green is added into the L-J matrix, and the mixture is packaged into test tubes after evenly mixed, 9-15 ml of each tube is baked and shaped at 80-90 ℃ for 30-50 min to obtain the Rogowski egg culture medium.
(2) Passaging of Sutong potato culture medium
Bacterial liquid inoculation or lawn coating methods are generally employed.
Inoculating bacterial liquid: in a biosafety cabinet, taking thalli meeting passage conditions on a Rogowski egg culture medium for 21-23 days, adding 1.0-3.0 mL of 0.9% sodium chloride injection on each Rogowski egg culture medium, washing the thalli according to a sterile operation mode, grinding to prepare a bacterial suspension, adjusting the concentration of the bacterial suspension to 80-100 mg/mL, inoculating the bacterial suspension into a Sutong potato culture medium at 0.4 mL/branch, and standing and culturing for 12-18 days at the temperature of 37-39 ℃. Observing the growth state of the thalli, sampling for bacterial examination (acid-fast staining), and storing the strains on a culture medium of the storax potato for 2 months at the temperature of 2-8 ℃.
The preparation method of the culture medium for the Sutong and the potato culture medium for the Sutong comprises the following steps:
the preparation and use amount of the 1L of the storax culture medium are as follows: 0.5g of magnesium sulfate, 0.5g of dipotassium hydrogen phosphate, 2g of citric acid, 0.05g of ferric ammonium citrate, 60ml of glycerin, 12g of sodium glutamate, 0.5ml of 1% zinc sulfate and water to 1000ml; mixing the above culture medium, adjusting pH to 7.0-7.5, packaging into Lu's bottle, sterilizing at 121deg.C for 30 min.
The culture medium is prepared by packaging culture medium into potato tubes, adding potato blocks (semi-cylindrical shape with diameter of about 2cm and length of 4 cm) soaked in water for injection, 1% sodium carbonate and pH7.2-7.4 culture medium, and sterilizing at 121deg.C for 30 min.
The culture medium for the fierce and the culture medium for the fierce potato used in the embodiment of the invention can be prepared according to the method.
And (3) lawn coating: in a biosafety cabinet, taking thalli meeting passage conditions on a culture medium of the Sutong potato, culturing for 12-18 days, taking a lawn by an inoculating shovel, inoculating the lawn onto the culture medium of the Sutong potato, and inoculating 0.48-0.72 cm for each culture medium 2 The lawn is placed in a culture room for static culture for 12 to 18 days at the temperature of 37 to 39 ℃. Observing the growth state of the bacterial cells, sampling for bacterial examination (acid-fast staining), wherein the strain on the culture medium of the Sutong potato can be in the range of 2 to moreStoring for 2 months at 8 ℃.
(3) Passage of culture medium bacterial membrane
Taking the bacterial cells which are cultured for 12-18 days and meet the passage conditions on a culture medium (or a culture medium of the Sutong), shoveling a bacterial film by an inoculation shovel, inoculating the bacterial film on the culture medium of the Sutong, and inoculating 3.6-5.4 cm of each bottle of culture medium 2 The bacterial film is placed in a culture room for static culture for 8 to 12 days at the temperature of 37 to 39 ℃. The growth state of the cells was observed, and samples were taken for cell detection (acid-fast staining).
(4) Sutong culture medium yarn membrane passage
And taking thalli meeting passage conditions on a storax culture medium for 8-12 days, pressing the thalli to dryness, preparing a fungus suspension, and inoculating the fungus suspension into the storax culture medium. Inoculating 2-4 mL of bacterial suspension to each bottle of culture medium. Placing in a culture room at 37-39 ℃ for static culture for 12-18 days. Observing the growth state of the bacterial cells, sampling for bacterial examination (acid-fast staining), and storing the yarn film on a storax culture medium for 6 weeks at the temperature of 2-8 ℃.
Example 2: cell collection
And (3) taking bacterial cells which are cultured on the storax medium for 8-12 days in the example 1, passaging and meet the passaging conditions, transferring the bacterial cells into a bacterial cell collector through aseptic operation, pressing the bacterial cells to dry, and weighing the bacterial cells.
Example 3: after the thalli are dispersed, semi-finished products are prepared
Weighing corresponding 1 unit weight of thalli in the example 2 according to the table 1, respectively putting the thalli into a rotary bottle with 1000g of steel balls, grinding, adding a freeze-drying protective agent for dispersion, and uniformly mixing for 10 minutes to obtain a BCG vaccine stock solution for treatment; the stock solution of BCG vaccine for treatment, such as 9 units, 10 units, 13 units, 18 units, 20 units, etc. is prepared by respectively adding 1 unit weight of bacterial cells with integer times into corresponding rotary bottles, grinding, dispersing with protective agent, and mixing.
Sampling and detecting the concentration; the inspection of pure bacteria is carried out by a general rule 1101 aseptic inspection method (direct inoculation method) in the 2020 edition of the pharmacopoeia of the people's republic of China, and the growths are subjected to smear microscopic examination and are sterile.
Weighing the freeze-drying protective agent, adding the freeze-drying protective agent into the BCG stock solution for treatment, dispersing the BCG stock solution for treatment, mixing for 10 minutes to obtain a BCG semi-finished product for treatment, and sampling for concentration detection; the inspection of pure bacteria is carried out by a general rule 1101 aseptic inspection method (direct inoculation method) in the 2020 edition of the pharmacopoeia of the people's republic of China, and the growths are subjected to smear microscopic examination and are sterile. And (5) sub-packaging and freeze-drying.
TABLE 1 parameter table for preparing semi-finished products by thallus dispersion
Figure BDA0003976257100000091
Figure BDA0003976257100000101
The dispersion control 1-3 are samples to be subpackaged into penicillin bottles, and are integer times of unit thalli in the dispersion 1-3 respectively, the unit thalli with unit weight are respectively put into corresponding rotary bottles, and are ground, dispersed by a protective agent and then combined to obtain the stock solution of the BCG vaccine for treatment, and the reserved stock solution is 1/2 integer times of the unit stock solution.
Example 4 lyophilization of semi-finished product
4.1 according to the cell dispersion method in example 2, the semi-finished product prepared by dispersing cells according to the relevant parameters in "dispersion 1" in table 1 is filled into penicillin bottles within 4 hours, 1mL of each bottle is filled, the temperature of a baffle plate is controlled to be not higher than-40 ℃, after the filling is finished, the semi-finished product is automatically half-packed by a machine and then is sent into a freeze-drying box, after all the packaged samples enter the freeze-drying box, a box door is closed, the temperature is kept for not more than 2 hours, and freeze-drying treatment is carried out according to the following procedures:
the freeze-drying treatment comprises an annealing stage, a sublimation drying stage and an analysis drying stage, wherein the following parameters are controlled in the process:
and (3) an annealing stage: setting the temperature of the partition plate to be minus 10 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 6.5 hours. Then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 5 hours after the time for reaching the final temperature is not more than 0.5 hours; before annealing, the semi-finished product needs to be pre-frozen at the temperature of-40 ℃;
Sublimation drying stage: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 4 hours; continuously adjusting the temperature of the partition plate to-16 ℃, keeping the temperature for 6 hours, wherein the time for reaching the final temperature is 3 hours; the vacuum degree is controlled at 10pa;
and (3) analysis and drying: the temperature of the partition plate is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 5 hours; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 2.5h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree of the process to be 10pa, preserving the heat for 4h, and controlling the vacuum degree of the heat preservation process to be not higher than 5pa.
The BCG vaccine freeze-dried product for treatment is obtained.
4.2 control 1: according to the thallus dispersing method in the embodiment 2, the samples to be split and prepared after thallus dispersing according to the relevant parameters in the dispersing control 1 in the table 1 are respectively filled into penicillin bottles within 4 hours, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃ at the same time, after the filling is finished, the samples are automatically half-packed by a machine and then are sent into a freeze-drying box, after the split and packaged samples are all put into the freeze-drying box, the box door is closed, the temperature is kept for not more than 2 hours, and freeze-drying treatment is carried out according to the freeze-drying process in 4.1, so that the BCG vaccine freeze-dried product for treatment is obtained.
EXAMPLE 5 lyophilization of semi-finished product
5.1 according to the thallus dispersing method in example 2, the semi-finished product prepared by dispersing thallus according to the relevant parameters in 'dispersing 2' in table 1 is filled into penicillin bottles in 4h, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃, after the filling is finished, the plate layer is automatically half-packed by a machine and then is sent into a freeze-drying box, after all samples are filled into the freeze-drying box, a box door is closed, the temperature is kept for not more than 2h, and freeze-drying treatment is carried out according to the following procedures:
the freeze-drying treatment comprises an annealing stage, a sublimation drying stage and an analysis drying stage, wherein the following parameters are controlled in the process:
and (3) an annealing stage: setting the temperature of the partition plate to be-12 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 3 hours; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 4 hours after the time for reaching the final temperature is not more than 0.5 hour; before annealing, the semi-finished product needs to be pre-frozen at the temperature of-40 ℃;
sublimation drying stage: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 5 hours; continuously adjusting the temperature of the partition plate to-16 ℃, keeping the temperature for 11 hours, wherein the time for reaching the final temperature is 2.5 hours; the vacuum degree is controlled at 20pa;
And (3) analysis and drying: the temperature of the partition plate is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 3h; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 1h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree of the process to 20pa, preserving heat for 9h, and controlling the vacuum degree of the heat preservation process to be not higher than 5pa;
the BCG vaccine freeze-dried product for treatment is obtained.
5.2 control 2: according to the thallus dispersing method in the embodiment 2, the samples to be split and prepared after thallus dispersing according to the relevant parameters in the dispersing control 2 in the table 1 are respectively filled into penicillin bottles within 4 hours, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃ at the same time, after the filling is finished, the samples are automatically half-packed by a machine and then are sent into a freeze-drying box, after the split and packaged samples are all put into the freeze-drying box, the box door is closed, the temperature is kept for not more than 2 hours, and freeze-drying treatment is carried out according to the freeze-drying process in 5.1, so that the BCG vaccine freeze-dried product for treatment is obtained.
EXAMPLE 6 lyophilization of semi-finished product
6.1 according to the thallus dispersing method in example 2, the semi-finished product prepared by dispersing thallus according to the relevant parameters in 'dispersing 3' in table 1 is filled into penicillin bottles in 4h, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃, after the filling is finished, the semi-finished product is automatically half-packed by a machine and then is sent into a freeze-drying box, after all the sub-packed samples enter the freeze-drying box, a box door is closed, the temperature is kept for not more than 2h, and freeze-drying treatment is carried out according to the following procedures:
The freeze-drying treatment comprises an annealing stage, a sublimation drying stage and an analysis drying stage, wherein the following parameters are controlled in the process:
and (3) an annealing stage: setting the temperature of the partition plate to be-14 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 1 hour; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 3 hours after the time for reaching the final temperature is not more than 0.5 hour; before annealing, the semi-finished product needs to be pre-frozen at the temperature of-40 ℃;
sublimation drying stage: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to gradually rise to-20 ℃, and the time for reaching the final temperature is 260min; continuously adjusting the temperature of the partition plate to-16 ℃, keeping the temperature for 7h, wherein the time for reaching the final temperature is 160 min; the vacuum degree is controlled at 15pa;
and (3) analysis and drying: the temperature of the partition plate is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 3h; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 1.5h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree of the process to 15pa, preserving heat for 6h, and controlling the vacuum degree of the heat preservation process to be not higher than 5pa;
The BCG vaccine freeze-dried product for treatment is obtained.
6.2 control 3: according to the thallus dispersing method in the embodiment 2, the samples to be split and prepared after thallus dispersing according to the relevant parameters in the dispersing control 3 in the table 1 are respectively filled into penicillin bottles within 4 hours, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃ at the same time, after the filling is finished, the samples are automatically half-packed by a machine and then are sent into a freeze-drying box, after the split and packaged samples are all put into the freeze-drying box, the box door is closed, the temperature is kept for not more than 2 hours, and freeze-drying treatment is carried out according to the freeze-drying process in 6.1, so that the BCG vaccine freeze-dried product for treatment is obtained.
6.3 control 4: relates to a freeze drying technology for producing BCG vaccine which is accepted in industry, wherein a semi-finished product prepared by performing thallus dispersion according to relevant parameters in 'dispersion 4' in table 1 is filled into penicillin bottles in 4h according to a thallus dispersion method in an embodiment 2, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃ at the same time, after filling, the bottles are automatically half-packed by a machine and then are sent into a freeze-drying box, after all samples are filled into the freeze-drying box, a box door is closed, and the temperature is kept for not more than 2h, and freeze-drying treatment is performed according to the following procedures, including a freezing stage, a sublimation drying stage and an analytical drying stage.
The following parameters were controlled during lyophilization:
freezing: preserving heat for 4 hours under the condition that the temperature of the partition board is not higher than-40 ℃;
sublimation drying stage: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 260min; continuously adjusting the temperature of the partition plate to-16 ℃, keeping the temperature for 7h, wherein the time for reaching the final temperature is 160 min; the vacuum degree is controlled at 15pa;
and (3) analysis and drying: the temperature of the partition plate is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 3h; regulating the temperature of the separator to 15 ℃ at a speed of 15 ℃/h, and preserving heat for 1.5h; heating to 39 ℃ at a speed of 24 ℃/h, and controlling the vacuum degree in the process to 15pa; preserving heat for 6 hours, wherein the vacuum degree in the heat preservation process is not higher than 5pa;
the BCG vaccine freeze-dried product for treatment is obtained.
6.4 control 5: according to the thallus dispersion method in the embodiment 2, the semi-finished product prepared by performing thallus dispersion according to the relevant parameters in the 'dispersion 5' in the table 1 is filled into penicillin bottles in 4h, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be above the temperature of-9 ℃ above the eutectic point, after filling, the plate layer is automatically and semi-plugged by a machine and then is sent into a freeze-drying box, after the sample is fully filled into the freeze-drying box, a box door is closed, and the heat preservation is carried out for not more than 2h, so that the product is in a supercooled state before reaching the temperature of the eutectic point, the temperature of a bottom layer and the temperature of the surface layer are basically consistent, and cold accumulation is carried out; comprises a prefreezing stage, a sublimation drying stage and a resolution drying stage.
Pre-freezing: regulating the temperature of the partition plate to be below the eutectic point to-12 ℃, cooling the product at the speed of 1 ℃/h, and preserving the temperature for 3 hours; regulating the temperature of the partition plate to be not higher than-40 ℃, continuously cooling the product, keeping the temperature for 3 hours after the time for reaching the final temperature is not more than 0.5 hour;
sublimation drying stage: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 260min; continuously adjusting the temperature of the partition plate to-16 ℃, keeping the temperature for 7h, wherein the time for reaching the final temperature is 160 min; the vacuum degree is controlled at 15pa;
and (3) analysis and drying: the temperature of the partition plate is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 3h; regulating the temperature of the separator to 15 ℃ at a speed of 15 ℃/h, and preserving heat for 1.5h; heating to 39 ℃ at a speed of 24 ℃/h, controlling the vacuum degree in the process at 15pa, preserving heat for 6h, and controlling the vacuum degree in the heat preservation process at not higher than 5pa;
the freeze-drying treatment is carried out according to the process, so that the BCG vaccine freeze-dried product for treatment is prepared, has a white loose appearance, can be well molded, and has poor re-solubility.
EXAMPLE 7 lyophilization of semi-finished product
According to the thallus dispersing method in example 2, the semi-finished product prepared by performing thallus dispersion according to the relevant parameters in 'dispersion 6' in table 1 is filled into penicillin bottles in 4h, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃ at the same time, after the filling is finished, the semi-finished product is automatically half-plugged by a machine and then is sent into a freeze-drying box, after all the samples are packaged, the box door is closed, the temperature is kept for not more than 2h, and freeze-drying treatment is performed according to the following procedures.
The following parameters were controlled during lyophilization:
and (3) an annealing stage: setting the temperature of the partition plate to be-14 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 5 hours; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 3 hours after the time for reaching the final temperature is not more than 0.5 hour; before annealing, the semi-finished product needs to be pre-frozen at the temperature of-40 ℃;
sublimation drying stage: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 4 hours; continuously adjusting the temperature of the partition plate to-16 ℃, keeping the temperature for 6 hours, wherein the time for reaching the final temperature is 2.5 hours; the vacuum degree is controlled at 10pa;
and (3) analysis and drying: the temperature of the partition plate is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 3h; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 1h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree of the process to be 10pa, preserving heat for 4h, and controlling the vacuum degree of the heat preservation process to be not higher than 5pa;
the BCG vaccine freeze-dried product for treatment is obtained.
EXAMPLE 8 lyophilization of semi-finished product
According to the thallus dispersing method in example 2, the semi-finished product prepared by performing thallus dispersion according to the relevant parameters of 'dispersion 7' in table 1 is filled into penicillin bottles within 4 hours, 1mL of each bottle is filled, the temperature of a plate layer is controlled to be not higher than-40 ℃, after the filling is finished, the plate layer is automatically half-plugged by a machine and then is sent into a freeze-drying box, after all samples are filled into the freeze-drying box, a box door is closed, the temperature is kept for not more than 2 hours, and freeze-drying treatment is performed according to the following procedures.
The following parameters were controlled during lyophilization:
and (3) an annealing stage: setting the temperature of the partition plate to be-14 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 5 hours; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 5 hours after the time for reaching the final temperature is not more than 0.5 hours; before annealing, the semi-finished product needs to be pre-frozen at the temperature of-40 ℃;
sublimation drying stage: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 5 hours; continuously adjusting the temperature of the partition plate to-16 ℃ and keeping the temperature for 11 hours, wherein the time for reaching the final temperature is 3 hours; the vacuum degree is controlled at 20pa;
and (3) analysis and drying: the temperature of the partition plate is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 5 hours; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 2.5h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree of the process to 20pa, preserving heat for 9h, and controlling the vacuum degree of the heat preservation process to be not higher than 5pa;
the BCG vaccine freeze-dried product for treatment is obtained.
Test example 1 BCG quality evaluation
The semi-finished products of examples 4 to 8, 6 to 7 cells and the final product of BCG for treatment prepared by freeze-drying (examples 4 to 8), the semi-finished products of comparative examples 1 to 3 cells and the final product of BCG for treatment prepared by freeze-drying (comparative examples 1 to 3), the semi-finished products of comparative examples 4 to 5 cells and the final product of BCG for treatment prepared by freeze-drying (comparative examples 4 and 5) were sampled and tested, the measurement methods and standards were found in the three parts of the pharmacopoeia of the people's republic of China, and the measurement results were found in Table 2 below. The test results show that the viable count and the thermal stability of the freeze-dried products (the viable count is basically unchanged after being placed for 28 days at the temperature of 2-8 ℃) of the examples 4-8 are higher than those of the dispersion control (controls 1-3) and the control freeze-drying process (controls 4 and 5). The quality is far higher than the standard required by the three parts of pharmacopoeia of the people's republic of China, and the stability is good. The results show that the semi-finished products prepared by dispersing the thalli in examples 4-8 and the finished products of the BCG vaccine for treatment prepared by freeze-drying can reduce the death rate of bacteria in the freeze-drying process, are suitable for being stored under a cold chain piece, and are safe. Viable count unit: 10 6 CFU/mg。
TABLE 2
Figure BDA0003976257100000161
Test example 2 cell immunity Effect test of BCG vaccine lyophilized product
The BCG vaccine freeze-dried product (example 4), the control dispersion method (control 1) and the control freeze-drying process (control 4) for treatment are respectively prepared at the concentration of 10mg/mL, 5mg/mL and 2.5mg/mL and the concentration of 1 multiplied by 10 7 The MB49 cell suspensions of each group were mixed in equal volumes, female C57BL/6J mice were transplanted subcutaneously, and the model control group was transplanted with only MB49 cell suspensions of corresponding concentrations, 10 animals each, 0.1mL each, 3 weeksThe animals are sacrificed later to measure the weight of solid tumors, the tumor growth inhibition rate is calculated, and the tumor inhibition effect and the cellular immunity evaluation of the MB49 tumor cells by the BCG vaccine for treatment are carried out.
Animal anatomy: after 3 weeks of inoculation, all groups of mice were euthanized, the spleens of the animals were isolated, and spleen single lymphocyte suspensions were prepared. Dissecting subcutaneous complete tumor and weighing; tumor growth inhibition (%) was calculated for each group.
(tumor growth inhibition (%) = (weight of model control group tumor-weight of test group tumor)/weight of model control group tumor x 100%).
ELISPOT method to detect secreted antigen specific IFN-gamma, IL-2 cell spot number: adjusting spleen lymphocyte concentration to 2.5X10 6 Mu.l of cell suspension was added per well, BCG-PPD was used as in vitro stimulator (50. Mu.l/well), conA (50. Mu.l/well) was used as positive control, DMEM (50. Mu.l/well) was used as negative control, all stimulators were multiplexed, and then at 37℃and 5% CO 2 Culturing in an incubator for 12-48 h, developing, and counting the spot forming cell number (SFC) by adopting an enzyme-linked spot counter.
And (3) data processing: data analysis was performed using Minitab software, using a double sample T test for significance of differences between tumor weight groups, and single factor analysis of variance (onegaanova) for significance of differences between formation of the cell spot number (SFC) groups, with P <0.05 indicating significance of differences. The results are shown in Table 3. The test result shows that the cell number of the embodiment 4 is obviously higher than that of the control dispersion (control 1) and the control freeze-drying process (control 4) in the secretion of BCG-PPD specific IFN-gamma and IL-2, and the cell immune effect is excellent, so that the cell dispersion method and the freeze-drying process can be used for preparing BCG vaccine products for treatment.
TABLE 3 secretion of BCG-PPD specific IFN-gamma, IL-2 cell count (SFC/2.5E5, n=10)
Figure BDA0003976257100000171
Figure BDA0003976257100000181
Table 3 notes: p (P) a Mean that compared to model control,: indicating significant level of difference (0.01<P<0.05),**: indicating a very significant level of difference (P <0.01 A) is provided; p: representation of delta compared to control: indicating significant level of difference (0.01<P<0.05 A) and ΔΔ: indicating a very significant level of difference (P<0.01)。
Test example 3 stability Effect test of BCG vaccine lyophilized products
The semi-finished products obtained by dispersing the thalli of the examples 4 to 8 and freeze-drying the semi-finished products are respectively placed in environments of 5+/-3 ℃, 25+/-2 ℃ and 37+/-2 ℃ to perform long-term stability study at normal storage temperature (5+/-3 ℃) on one hand, and the study results are shown in Table 4:
TABLE 4 results of Long-term stability study of BCG vaccine products for treatment (5 ℃ + -3 ℃)
Figure BDA0003976257100000182
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Figure BDA0003976257100000191
On the other hand, considering the risks of the product during storage and transportation and during use, stability studies under different temperatures (25 ℃ ± 2 ℃ and 37 ℃ ± 2 ℃) were carried out, and the results are shown in table 5:
TABLE 5 results of Long-term stability study of BCG vaccine products for treatment (37 ℃ + -3 ℃)
Figure BDA0003976257100000201
As can be seen from the data in tables 4 and 5, the BCG vaccine product for treatment prepared by adopting the specific thallus dispersion method and the freeze-drying process is placed in an environment of 5+/-3 ℃ for 24 months, an environment of 25+/-2 ℃ for 6 months and an environment of 37+/-2 ℃ for 3 months, has stable quality, meets the requirements, can effectively prolong the effective period, prolongs the effective period from 18 months to 24 months, and is suitable for storage, transportation and use under normal temperature and high temperature conditions.
Application example 1 the preparation is carried out by adopting the dispersion method of the thalli and the freeze drying method of the BCG semi-finished product, and the production conditions of each batch of the BCG preparation for treatment are shown in the table 6:
TABLE 6
Figure BDA0003976257100000211
Although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (14)

1. A method for dispersing bacillus calmette-guerin bacteria for treatment is characterized in that,
15 g-20 g of thalli are dispersed into 125 g-167 g of freeze-drying protective agent, and 1 unit weight of bacillus calmette-guerin vaccine stock solution for treatment with the concentration of bacterial suspension of 120 mg/mL-140 mg/mL is obtained;
dispersing the BCG vaccine stock solution for treatment by 1.8-2.5 times by adopting a freeze-drying protective agent, and uniformly mixing to obtain a BCG vaccine semi-finished product for treatment with 1 unit weight;
the concentration of the BCG bacterium in the 1 unit weight of the semi-finished product of the BCG vaccine for treatment is 48-72 mg/mL.
2. The method for dispersing bacillus calmette-guerin bacteria of claim 1 wherein,
The freeze-drying protective agent comprises 10% of sucrose, 1% of gelatin, 1% of sodium glutamate, 1% of potassium chloride and the balance of water for injection.
3. The method for dispersing bacillus calmette-guerin bacteria of claim 1 wherein,
the BCG vaccine stock solution for treatment comprises the following steps:
grinding 15 g-20 g BCG strain according to the mass ratio of the BCG strain to 1000g steel balls;
and (3) grinding, adding 125-167 g of freeze-drying protective agent, and uniformly mixing for 10 minutes to obtain the stock solution of the BCG vaccine for treatment.
4. The method for dispersing bacillus calmette-guerin bacteria of claim 3,
the grinding conditions are as follows: the rotating speed is 180-200 r/min, and the time is 3-5 min.
5. The method for dispersing bacillus calmette-guerin bacteria of any one of claims 1 to 4,
the mass ratio of the bacillus calmette-guerin bacteria to the freeze-drying protective agent is 17-20 g:125 g-135 g.
6. The method for dispersing bacillus calmette-guerin bacteria of any one of claims 1 to 4,
the mass ratio of the bacillus calmette-guerin thallus to the freeze-drying protective agent is 19.8g:130g.
7. The method for dispersing bacillus calmette-guerin bacteria of any one of claims 1 to 4,
The concentration of the BCG bacterium in the 1 unit weight of the semi-finished product of the BCG vaccine for treatment is 54-66 mg/mL.
8. The method for dispersing bacillus calmette-guerin bacteria of any one of claims 1 to 4,
the concentration of the BCG bacterium in the 1 unit weight of the semi-finished product of the BCG vaccine for treatment is 60mg/mL.
9. The therapeutic bcg stock solution of claim 1, wherein said therapeutic bcg stock solution has a weight 1-120 times the weight of 1 unit of said bcg stock solution.
10. The therapeutic bcg intermediate of claim 1 wherein said therapeutic bcg intermediate has a weight of 1 to 120 times the weight of said therapeutic bcg intermediate.
11. A method of lyophilizing a semi-finished product of BCG for therapeutic use as defined in claim 1,
and (3) putting the semi-finished product of the BCG vaccine for treatment into a freeze-drying box under the precooling condition of less than or equal to minus 40 ℃, preserving heat for not more than 2 hours after all the semi-finished products are put into the box, sequentially carrying out annealing, sublimation drying and analysis drying, and pressing a plug and rolling a cover after the semi-finished product of the BCG vaccine for treatment is finished to obtain the finished product of the BCG vaccine for treatment.
12. The method of lyophilization of a therapeutic bcg vaccine blank according to claim 11,
The annealing is as follows: setting the temperature of the partition plate to be any one of-10 to-14 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 1 to 6.5 hours; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 3-5 h, wherein the time for reaching the final temperature is not more than 0.5 h;
the sublimation drying is as follows: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 30pa, the temperature of the partition plate is regulated to be increased to-20 ℃, and the time for reaching the final temperature is 4-5 hours; continuously adjusting the temperature of the partition plate to-16 ℃ and keeping the temperature for 6-11 hours, wherein the time for reaching the final temperature is 2.5-3 hours; the vacuum degree is controlled to be 10-20 pa;
the analysis and drying stage is as follows: the temperature of the partition board is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching the final temperature is 3-5 h; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 1-2.5 h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree in the process at 10-20 pa, and preserving the heat for 4-9 h, wherein the vacuum degree in the heat preservation process is not higher than 5pa.
13. The method for lyophilization of therapeutic bcg vaccine semi-finished product according to claim 11 or 12, characterized in that,
the annealing is as follows: setting the temperature of the partition board to be minus 14 ℃ for heating, wherein the time for reaching the final temperature is 3 hours, and preserving the heat for 1 to 6.5 hours; then setting the temperature of the partition plate to be not higher than-40 ℃ for cooling, keeping the temperature for 3-5 h, wherein the time for reaching the final temperature is not more than 0.5 h;
The sublimation drying is as follows: when the temperature of the condenser is reduced to be lower than-50 ℃, vacuumizing is started, when the vacuum degree is reduced to 10-20 pa, the temperature of the partition plate is regulated to be gradually increased to-20 ℃, and the time for reaching the final temperature is 4-5 h; continuously adjusting the temperature of the partition plate to-16 ℃ and keeping the temperature for 6-11 hours, wherein the time for reaching the final temperature is 2.5-3 hours; the vacuum degree is controlled to be 10-20 pa;
the analysis and drying stage is as follows: the temperature of the partition board is regulated to gradually rise from-16 ℃ to 0 ℃ and the time for reaching 0 ℃ is 3-5 h; continuously regulating the temperature of the partition plate to 15 ℃ at the speed of 15 ℃/h, and preserving heat for 1-2.5 h; continuously heating to 39 ℃ at the speed of 24 ℃/h, controlling the vacuum degree in the process at 10-20 pa, and preserving the heat for 4-9 h, wherein the vacuum degree in the heat preservation process is not higher than 5pa.
14. A finished therapeutic bcg vaccine obtained by the lyophilization process of any one of claims 11-13.
CN202211538816.0A 2022-12-01 2022-12-01 Dispersing and freeze-drying method for bacillus calmette-guerin thallus for treatment, stock solution, semi-finished product and finished product Pending CN116077634A (en)

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