阅读说明：本技术 一种改良型的冻干甲型肝炎减毒活疫苗稳定剂、疫苗半成品、疫苗成品及其制备方法 (Improved freeze-dried live attenuated hepatitis A vaccine stabilizer, vaccine semi-finished product, vaccine finished product and preparation method thereof ) 是由 徐艳玲 刘令九 侯丽娟 王艺博 顾建阳 周延彬 *** 李育红 鞠闯 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种改良型的冻干甲型肝炎减毒活疫苗稳定剂,包括以下组分和浓度：海藻糖：2％-6％,谷氨酸钠：0.1％-1.0％,精氨酸：0.05％-0.3％,尿素：0.1％-0.8％,维生素C：0％-0.5％,右旋糖酐40：0％-2.0％,山梨醇：0％-0.5％,甘露醇：0.05％-0.5％,余量为注射用水。本发明提供了一种改良型的冻干甲型肝炎减毒活疫苗稳定剂,通过减少稳定剂中药品,不仅增加了产品的溶解速度,而且减少了机体潜在的过敏反应。本发明还提供了一种改良型的冻干甲型肝炎减毒活疫苗成品的制备方法,使用改良稳定剂进行半成品制备成品,不仅可以简化操作步骤,降低物料使用成本,而且减少染菌风险,提高产品质量。(The invention discloses an improved freeze-dried hepatitis A attenuated live vaccine stabilizer, which comprises the following components in concentration: trehalose: 2% -6%, sodium glutamate: 0.1% -1.0%, arginine: 0.05% -0.3%, urea: 0.1% -0.8%, vitamin C: 0% -0.5%, dextran 40: 0% -2.0%, sorbitol: 0% -0.5%, mannitol: 0.05 to 0.5 percent of the total weight of the injection and the balance of water for injection. The invention provides an improved freeze-dried hepatitis A attenuated live vaccine stabilizer, which not only increases the dissolution speed of a product, but also reduces the potential anaphylactic reaction of an organism by reducing medicines in the stabilizer. The invention also provides a preparation method of the improved freeze-dried live attenuated hepatitis A vaccine finished product, and the improved stabilizer is used for preparing the semi-finished product, so that the operation steps can be simplified, the material use cost can be reduced, the bacterial contamination risk can be reduced, and the product quality can be improved.)

1. An improved freeze-dried live attenuated hepatitis A vaccine stabilizer is characterized by comprising the following components in percentage by mass and volume:

trehalose: 2% -6%, sodium glutamate: 0.1% -1.0%, arginine: 0.05% -0.3%, urea: 0.1% -0.8%, vitamin C: 0% -0.5%, dextran 40: 0% -2.0%, sorbitol: 0% -0.5%, mannitol: 0.05 to 0.5 percent of the total weight of the injection and the balance of water for injection.

2. A modified lyophilized hepatitis A attenuated live vaccine semi-finished product, characterized in that, the modified lyophilized hepatitis A attenuated live vaccine stabilizer of claim 1 is used for preparation.

3. A method for preparing a modified lyophilized live attenuated hepatitis a vaccine semi-finished product, which is used for preparing the modified lyophilized live attenuated hepatitis a vaccine semi-finished product of claim 2, comprising the following steps:

step one, purifying a hepatitis A virus harvest solution to obtain a vaccine stock solution;

step two, preparing the improved freeze-dried hepatitis A attenuated live vaccine stabilizer;

step three, calculating and measuring a diluent;

and step four, mixing the vaccine stock solution, the vaccine stabilizer and the diluent uniformly in sequence to obtain a semi-finished product.

4. The improved freeze-dried hepatitis A attenuated live vaccine semi-finished product of claim 3, wherein in the second step, the process of preparing the improved freeze-dried hepatitis A attenuated live vaccine stabilizer comprises the following steps:

step 1, preparing an initial vaccine stabilizer;

step 2, determining the liquid amount of a semi-finished product according to the liquid amount of the vaccine stock solution and the virus titer, calculating the liquid amount of the improved freeze-dried hepatitis A attenuated live vaccine stabilizer according to 50% of the liquid volume of the semi-finished product, and measuring;

wherein, the improved freeze-dried hepatitis A attenuated live vaccine stabilizer comprises the following components in percentage by mass:

trehalose: 2% -6%, sodium glutamate: 0.1% -1.0%, arginine: 0.05% -0.3%, urea: 0.1% -0.8%, vitamin C: 0% -0.5%, dextran 40: 0% -2.0%, sorbitol: 0% -0.5%, mannitol: 0.05 to 0.5 percent of the total weight of the injection and the balance of water for injection.

5. The improved process for preparing a semi-finished lyophilized live attenuated hepatitis A vaccine according to claim 4, wherein the liquid amount of the diluent is calculated in the third step by:

V₁＝V₀-V₂-V₃；

wherein, V₁Amount of dilution liquid, V₀Amount of liquid as a semi-finished product, V₂The amount of the stock solution of the vaccine, V₃Is the amount of vaccine stabilizer.

6. The improved process for preparing a semi-finished product of lyophilized live attenuated hepatitis A vaccine according to claim 5, wherein in step four, the vaccine stock solution, the vaccine stabilizer and the diluent are sequentially combined and shaken to obtain the semi-finished product.

7. An improved freeze-dried live attenuated hepatitis A vaccine finished product, which is characterized in that the improved freeze-dried live attenuated hepatitis A vaccine stabilizer of claim 1 is used for preparation.

8. A method for preparing the improved freeze-dried live attenuated hepatitis A vaccine, which is used for preparing the improved freeze-dried live attenuated hepatitis A vaccine of claim 7, comprising the following steps:

step one, obtaining an improved semi-finished product of the freeze-dried live attenuated hepatitis A vaccine;

and step two, freeze-drying the semi-finished product to prepare the improved freeze-dried live attenuated hepatitis A vaccine finished product.

9. The improved freeze-dried live attenuated hepatitis A vaccine as claimed in claim 8, wherein the freeze-drying process comprises:

keeping the temperature at minus 30 ℃ to minus 60 ℃ for 2 to 6 hours to obtain a pre-frozen semi-finished product;

placing the pre-frozen semi-finished product in a primary sublimation freeze-drying box with a clapboard at the temperature of-30 to-60 ℃ for primary freeze-drying to obtain a primary freeze-dried semi-finished product;

and (3) placing the primary freeze-dried semi-finished product into a secondary sublimation freeze-drying box with a partition plate at the temperature of 10-40 ℃ for secondary freeze-drying to obtain the improved freeze-dried hepatitis A attenuated live vaccine finished product.

10. The improved freeze-dried live attenuated hepatitis A vaccine finished product preparation method according to claim 8, characterized in that the improved freeze-dried live attenuated hepatitis A vaccine finished product has a size of 0.5 mL/bottle after reconstitution.

Technical Field

The invention relates to a freeze-dried live attenuated hepatitis A vaccine, in particular to an improved freeze-dried live attenuated hepatitis A vaccine stabilizer and a preparation method of a finished product.

Background

The freeze-dried hepatitis A attenuated live vaccine contains a certain amount of stabilizer, and has the functions of keeping the activity of target protein in a freeze-dried product and ensuring the uniformity, the smoothness and the stability of the surface of the freeze-dried product, wherein the vitamin C, the dextran 40 and the sorbitol are added into the vaccine as one of the stabilizer components to play the roles of shaping, supporting and the like.

At present, the vaccine stabilizers used by the freeze-dried live attenuated hepatitis A vaccine used by my company are as follows: the final trehalose concentration was: 2 to 6 percent, the final concentration of sodium glutamate is 0.1 to 1.0 percent, the final concentration of arginine is 0.05 to 0.3 percent, the final concentration of urea is 0.1 to 0.8 percent, the final concentration of vitamin C is 0.05 to 0.5 percent, the final concentration of dextran 40 is 3 to 5 percent, the final concentration of sorbitol is 0.05 to 0.5 percent, and the final concentration of mannitol is 0.05 to 0.5 percent.

However, the current technology has the following disadvantages:

1. vitamin C, dextran 40 and sorbitol are added into the vaccine as one of the components of the stabilizer, the types of the auxiliary materials used in the existing stabilizer are too many, when the concentrations of the three auxiliary materials in the vaccine are too high, the dissolution speed of the product is slow, potential anaphylactic reaction can be caused in an organism, and meanwhile, the production cost is increased due to the use of the too high auxiliary materials;

2. in the semi-finished product preparation stage, the stock solution needs to be mixed with a concentrated stabilizer I, a stabilizer II, a stabilizer III and the vaccine solution in sequence, the operation steps are complicated, and the risk of contamination is increased.

3. The original vaccine is 1.0 ml/bottle after redissolution, and has the following defects:

(1) the infant inoculation has a large pain feeling, and is not favorable for the popularization and the application of planned immunity.

(2) The injection dosage is 1.0ml, on the premise of achieving the same immune effect, the non-viral components (such as auxiliary material components) in the body of the vaccine inoculation are higher than the vaccine with the specification of 0.5ml after redissolution, and the probability of causing adverse reaction by the vaccine is possibly higher than the vaccine with the specification of 0.5 ml.

(3) Live virus may be spilled during vaccine injection.

(4) The injection dose of the vaccine is 1.0ml, and compared with the vaccine with the specification of 0.5ml, the dosage of the auxiliary materials is increased, so that the production cost is increased.

Disclosure of Invention

The invention provides an improved freeze-dried live attenuated hepatitis A vaccine stabilizer for solving the technical defects at present, which not only increases the dissolution speed of the product, but also reduces the potential anaphylactic reaction of organisms by reducing medicines in the stabilizer.

The invention also provides an improved freeze-dried hepatitis A attenuated live vaccine semi-finished product.

The invention also provides an improved freeze-dried live attenuated hepatitis A vaccine finished product.

The invention also provides a preparation method of the improved freeze-dried hepatitis A attenuated live vaccine semi-finished product, and the improved stabilizer is used for preparing the semi-finished product, so that the operation steps can be simplified, the material use cost is reduced, the bacterial contamination risk is reduced, and the product quality is improved.

The invention also provides a preparation method of the improved freeze-dried live attenuated hepatitis A vaccine finished product, and the improved freeze-drying operation of the finished product can simplify the operation steps, reduce the material use cost, reduce the risk of bacterial contamination and improve the product quality.

The technical scheme provided by the invention is as follows:

an improved freeze-dried live attenuated hepatitis A vaccine stabilizer, which comprises the following components in percentage by mass and volume:

trehalose: 2% -6%, sodium glutamate: 0.1% -1.0%, arginine: 0.05% -0.3%, urea: 0.1% -0.8%, vitamin C: 0% -0.5%, dextran 40: 0% -2.0%, sorbitol: 0% -0.5%, mannitol: 0.05 to 0.5 percent of the total weight of the injection and the balance of water for injection.

An improved freeze-dried attenuated live hepatitis A vaccine semi-finished product is prepared by using the improved freeze-dried attenuated live hepatitis A vaccine stabilizer.

A method for preparing an improved freeze-dried hepatitis A attenuated live vaccine semi-finished product, which is used for the improved freeze-dried hepatitis A attenuated live vaccine semi-finished product, comprises the following steps:

step one, purifying a hepatitis A virus harvest solution to obtain a vaccine stock solution;

step two, preparing the improved freeze-dried hepatitis A attenuated live vaccine stabilizer;

step three, calculating and measuring a diluent;

and step four, mixing the vaccine stock solution, the vaccine stabilizer and the diluent uniformly in sequence to obtain a semi-finished product.

Preferably, in the second step, the process of preparing the modified freeze-dried live attenuated hepatitis A vaccine stabilizer comprises the following steps:

step 1, preparing an initial vaccine stabilizer;

step 2, determining the liquid amount of a semi-finished product according to the liquid amount of the vaccine stock solution and the virus titer, calculating the liquid amount of the improved freeze-dried hepatitis A attenuated live vaccine stabilizer according to 50% of the liquid volume of the semi-finished product, and measuring;

wherein, the improved freeze-dried hepatitis A attenuated live vaccine stabilizer comprises the following components in percentage by mass:

trehalose: 2% -6%, sodium glutamate: 0.1% -1.0%, arginine: 0.05% -0.3%, urea: 0.1% -0.8%, vitamin C: 0% -0.5%, dextran 40: 0% -2.0%, sorbitol: 0% -0.5%, mannitol: 0.05 to 0.5 percent of the total weight of the injection and the balance of water for injection.

Preferably, in the third step, the method for calculating the liquid amount of the diluent includes:

V₁＝V₀-V₂-V₃；

wherein, V₁Amount of dilution liquid, V₀Amount of liquid as a semi-finished product, V₂The amount of the stock solution of the vaccine, V₃Is the amount of vaccine stabilizer.

Preferably, in the fourth step, the vaccine stock solution, the vaccine stabilizer and the diluent are combined in sequence, and the semi-finished product is obtained by mixing and oscillating simultaneously.

An improved freeze-dried live attenuated hepatitis A vaccine finished product is prepared by using the improved freeze-dried live attenuated hepatitis A vaccine stabilizer.

A method for preparing an improved freeze-dried live attenuated hepatitis A vaccine finished product, which is used for the improved freeze-dried live attenuated hepatitis A vaccine finished product, comprises the following steps:

step one, obtaining an improved semi-finished product of the freeze-dried live attenuated hepatitis A vaccine;

and step two, freeze-drying the semi-finished product to prepare the improved freeze-dried live attenuated hepatitis A vaccine finished product.

Preferably, the lyophilization method is:

keeping the temperature at minus 30 ℃ to minus 60 ℃ for 2 to 6 hours to obtain a pre-frozen semi-finished product;

placing the pre-frozen semi-finished product in a primary sublimation freeze-drying box with a clapboard at the temperature of-30 to-60 ℃ for primary freeze-drying to obtain a primary freeze-dried semi-finished product;

and (3) placing the primary freeze-dried semi-finished product into a secondary sublimation freeze-drying box with a partition plate at the temperature of 10-40 ℃ for secondary freeze-drying to obtain the improved freeze-dried hepatitis A attenuated live vaccine finished product.

Preferably, the modified freeze-dried live attenuated hepatitis A vaccine finished product has a specification of 0.5 mL/bottle after being reconstituted.

The invention has the following beneficial effects:

1) the use of the improved stabilizer can improve the stability of the vaccine and ensure the safety and relative efficacy of the vaccine. The reduction of the use amount of the traditional Chinese medicine for improving the stabilizer not only increases the dissolution speed of the product, reduces the potential anaphylactic reaction of the organism, but also reduces the production cost.

2) When the improved stabilizer is used for preparing semi-finished products, the operation steps can be simplified, the use cost of materials is reduced, the risk of bacterial contamination is reduced, and the product quality is improved.

3) The finished vaccine prepared by the semi-finished product has a specification of 0.5 ml/bottle after redissolution, the appearance of the vaccine, the detection result of visible foreign matter, water content, virus titer and thermal stability tests all accord with the regulations, the market development trend is complied with, the production cost is reduced, and higher economic benefit can be brought.

4) On the premise of ensuring that the immune effect and safety of the vaccine are not changed, the vaccine in 0.5 ml/bottle after redissolution can not only reduce the pain during inoculation, increase the compliance, but also prevent live virus from overflowing, and simultaneously expand the popularization and application of planned immunity.

Detailed Description

The present invention is described in further detail below to enable those skilled in the art to practice the invention with reference to the description.

The invention provides an improved freeze-dried attenuated hepatitis A vaccine stabilizer, which comprises the following specific formula (final components and concentration, the final concentration is mass volume fraction, g/mL): trehalose: 2% -6%, sodium glutamate: 0.1% -1.0%, arginine: 0.05% -0.3%, urea: 0.1% -0.8%, vitamin C: 0% -0.5%, dextran 40: 0% -2.0%, sorbitol: 0% -0.5%, mannitol: 0.05 to 0.5 percent of the total weight of the injection and the balance of water for injection.

The invention also provides a preparation method of the improved freeze-dried hepatitis A attenuated vaccine finished product, the improved stabilizer is prepared according to the formula, the liquid volume of the stabilizer is calculated by 50 percent of the liquid volume of the semi-finished product and is mixed with the stock solution diluent to form the semi-finished product, the preparation method of the semi-finished product is simple and convenient, the operation is easy, and the risk of bacterial contamination can be reduced. The specification of the semi-finished product after subpackaging, plugging and freeze-drying is 0.5 ml/bottle after redissolution, on the premise of ensuring that the immune effect is unchanged, the specification of the product after redissolution is 0.5 ml/bottle, compared with the specification of the product after redissolution of 1.0 ml/bottle, the pain feeling during inoculation is reduced, the probability of possible occurrence of adverse reaction is reduced, the possibility of overflowing of live viruses is prevented, and meanwhile, the production cost can be saved.

The preparation method of the improved freeze-dried attenuated hepatitis A vaccine mainly comprises the following steps:

step 1, preparing a hepatitis A virus harvest solution through an attenuated strain L-A-1 of a hepatitis A virus strain with the preservation number of CCTCC No. V92004, and then performing cell disruption and purification treatment after extraction on the virus harvest to obtain a vaccine stock solution.

Step 2, preparing a vaccine stabilizer, wherein the formula and the concentration (the final concentration is mass volume fraction, g/mL) are as follows (the concentrations of the components are 2 times of the concentrations of the corresponding improved stabilizer components): trehalose: 4% -12%, sodium glutamate: 0.2% -2.0%, arginine: 0.1% -0.6%, urea: 0.2% -1.6%, vitamin C: 0% -1.0%, dextran 40: 0% -4.0%, sorbitol: 0% -1.0%, mannitol: 0.1% -1.0%; the medicine is weighed according to the formula and then dissolved in water for injection, and the vaccine stabilizer is obtained after degerming and filtration.

Step 3, determining the liquid amount of the semi-finished product according to the liquid amount of the vaccine stock solution and the virus titer of the vaccine stock solution;

step 4, calculating and measuring the stabilizer: calculating the liquid amount of the improved stabilizer according to 50% of the liquid volume of the semi-finished product and measuring;

the improved stabilizer comprises the following specific formula (final components and concentration, the final concentration is mass volume fraction, g/mL): trehalose: 2% -6%, sodium glutamate: 0.1% -1.0%, arginine: 0.05% -0.3%, urea: 0.1% -0.8%, vitamin C: 0% -0.5%, dextran 40: 0% -2.0%, sorbitol: 0% -0.5%, mannitol: 0.05-0.5 percent of the total weight of the composition and the balance of water for injection

Step 5, calculating and measuring the diluent:

volume V of diluent₁Calculation method (unit mL):

V₁＝V₀-V₂-V₃

wherein, V₀The unit mL is the amount of the semi-finished liquid; v₂The amount of the vaccine stock solution is unit mL; v₃The unit mL is the amount of vaccine stabilizer.

Step 6, merging the semi-finished products: and sequentially introducing the vaccine stock solution, the stabilizer and the diluent into a special dilution and combination barrel, and combining and oscillating to obtain a semi-finished product.

And 7, filling the semi-finished product and then freeze-drying.

The specific freeze-drying method and conditions are as follows:

1) a pre-freezing stage: keeping the temperature between minus 30 ℃ and minus 60 ℃ for 2 to 6 hours;

2) primary freeze-drying: the temperature of a partition plate of the primary sublimation freeze-drying box is-30 to-60 ℃;

3) and (3) secondary freeze-drying: the temperature of the partition board of the secondary sublimation freeze-drying box is 10-40 ℃, thus obtaining the improved freeze-drying hepatitis A attenuated live vaccine finished product.

The improved freeze-dried live attenuated hepatitis A vaccine finished product is subjected to related detection, and the method comprises the following steps: appearance and visible foreign matter, moisture, virus titer, heat stability test. The result shows that all the detection results reach the qualified standard.

The invention is further illustrated by the following specific examples.