Vaccine freeze-drying protective agent, preparation and preparation method

Abstract

The present application relates to the field of biological products, and specifically to a vaccine freeze-dried protective agent, preparation, and preparation method. The freeze-dried protective agent comprises the following raw materials in parts by weight: 1-10 parts of arginine, 10-20 parts of sodium glutamate, 1-10 parts of isoleucine, 0.5-15 parts of glycine, 50-80 parts of trehalose, 10-50 parts of diethylaminoethyl dextran, and 20-60 parts of polyvinylpyrrolidone. The above components are dissolved in PBS solution and filtered. The freeze-dried protective agent is mixed with the vaccine to prepare a vaccine freeze-dried preparation. The components of the freeze-dried protective agent do not contain human or animal-derived ingredients, eliminating the potential risk of exogenous viral contamination and are safe and reliable. The freeze-dried preparation improves the stability of the vaccine during storage.

Description

Technical Field

[0001] The present application relates to the field of biological products, and specifically to a vaccine freeze-dried protective agent, preparation and preparation method. Background Art

[0002] Rabies is an acute zoonotic infectious disease caused by a virus of the genus Lyssavirus, typically leading to acute encephalitis or meningitis. Rabies vaccine and rabies immunoglobulin are the only effective means of preventing rabies.

[0003] Vaccines are commonly formulated for oral administration and injection. To ensure molecular activity, current vaccine formulation research focuses primarily on injections, and lyophilized formulations have become the primary injectable formulation due to their high stability. During the vacuum freeze-drying process, both freezing and drying inevitably cause protein denaturation. Therefore, lyoprotectants that simultaneously meet the three requirements of safety, stability, and efficacy have become a key research target for lyophilized biological products.

[0004] Most of the freeze-dried human rabies vaccines currently on the market contain human albumin as a key ingredient. Human albumin, derived from human plasma, is an excellent stabilizer for freeze-drying, effectively protecting the protein's activity. However, human albumin is expensive, increasing vaccine production costs. Furthermore, its complex composition, derived from human blood, can pose a hidden risk of vaccine contamination.

[0005] Therefore, a freeze-dried human rabies vaccine without human or animal components will be one of the development directions in the field of rabies prevention research, and the development of freeze-dried protective agents is the focus and difficulty of the research on serum-free human rabies vaccine. Summary of the Invention

[0006] In order to improve safety and stability, the present application provides a serum-free freeze-dried human rabies vaccine freeze-dried protectant, preparation and preparation method.

[0007] In the first aspect, the present application provides a serum-free freeze-dried human rabies vaccine freeze-dried protectant, which adopts the following technical solution:

[0008] A serum-free freeze-dried human rabies vaccine lyoprotectant comprises the following raw materials in parts by weight: 1-10 parts of arginine, 10-20 parts of sodium glutamate, 1-10 parts of isoleucine, 0.5-15 parts of glycine, 50-80 parts of trehalose, 10-50 parts of diethylaminoethyl dextran, and 20-60 parts of polyvinyl pyrrolidone.

[0009] Preferably, the lyoprotectant comprises the following raw materials in parts by weight: 1-7 parts of arginine, 10-16 parts of sodium glutamate, 5-10 parts of isoleucine, 0.5-10 parts of glycine, 50-80 parts of trehalose, 10-40 parts of diethylaminoethyl dextran, and 20-50 parts of polyvinyl pyrrolidone.

[0010] Preferably, the lyophilization protective agent comprises the following raw materials in parts by weight: 5 parts of arginine, 12 parts of sodium glutamate, 6 parts of isoleucine, 10 parts of glycine, 60 parts of trehalose, 20 parts of diethylaminoethyl dextran, and 40 parts of polyvinyl pyrrolidone.

[0011] In a second aspect, the present application provides a method for preparing a serum-free freeze-dried human rabies vaccine freeze-dried protectant, which adopts the following technical solution:

[0012] After each component is dissolved in PBS solution, sterile filtration is performed using a microporous filter membrane to obtain the product.

[0013] In a third aspect, the present application provides a freeze-dried vaccine formulation, which adopts the following technical solution:

[0014] A vaccine freeze-dried preparation is prepared by mixing the serum-free freeze-dried human rabies vaccine freeze-dried protective agent with the vaccine.

[0015] In a fourth aspect, the present application provides a method for preparing a freeze-dried vaccine formulation, which adopts the following technical solution:

[0016] A method for preparing a freeze-dried vaccine preparation comprises the following steps:

[0017] (1) mixing the vaccine with a lyophilizing protective agent to obtain a semi-finished product solution;

[0018] (2) The semi-finished product solution is pre-frozen, vacuumized, sublimated and dried, and desorbed to obtain a freeze-dried vaccine preparation.

[0019] Preferably, the weight ratio of the vaccine to the lyoprotectant is 1:(3-6). More preferably, the weight ratio is 1:4.

[0020] Preferably, the minimum temperature in the pre-freezing stage is -45-50°C, and is maintained for 2-4 hours after reaching the minimum temperature; and the vacuum pressure is 0.001-0.003 mbar.

[0021] Preferably, the final temperature in the sublimation drying stage is -30°C to -25°C, the time to reach the final temperature is 6-9 hours, and the final temperature is maintained for 5-7 hours after reaching the final temperature.

[0022] Preferably, the final temperature in the desorption drying stage is 25° C. to 30° C., the time to reach the final temperature is 3-5 hours, and the final temperature is maintained for 2-4 hours.

[0023] More preferred freeze-drying conditions are:

[0024] The lowest temperature in the pre-freezing stage is -45°C, which is maintained for 3 hours after reaching the lowest temperature; the vacuum pressure is 0.003mbar;

[0025] The final temperature of the sublimation drying stage was -29°C, the time to reach the final temperature was 7 h, and the final temperature was maintained for 6 h;

[0026] The final temperature of the desorption drying stage was 30°C, the time to reach the final temperature was 5 h, and the final temperature was maintained for 4 h.

[0027] In the above technical solution, diethylaminoethyl dextran can be used as a protein stabilizer, effectively replacing human serum albumin in related technologies, reducing the potential dangers brought by foreign viruses, and improving the stability of the vaccine.

[0028] Polyvinylpyrrolidone is a non-ionic polymer compound with excellent solubility and physiological compatibility. As a cosolvent or crystallization inhibitor in injection, it can play a good skeleton support role, reduce the residual moisture of freeze-dried preparations, and improve the stability of vaccines during storage.

[0029] The serum-free freeze-dried human rabies vaccine freeze-dried protectant provided in the present application does not contain any human or animal-derived components.

[0030] The matrix liquid used to prepare the freeze-dried protective agent of the present application is PBS buffer solution or water for injection.

[0031] In summary, this application has the following beneficial effects:

[0032] 1. This application provides a serum-free, freeze-dried, human rabies vaccine lyoprotectant. This protectant contains no human or animal-derived ingredients, eliminating the potential risk of exogenous viral contamination. Furthermore, the protectant provides excellent protection for the vaccine. Also provided is a method for using the serum-free, freeze-dried, human rabies vaccine lyoprotectant. This method is quick to use and significantly reduces costs.

[0033] 2. The present application provides a serum-free, freeze-dried human rabies vaccine freeze-dried formulation. The freeze-dried formulation has low residual moisture, improving the vaccine's stability during storage. Compared to related technologies, the freeze-dried vaccine formulation provided in this application eliminates the risk of exogenous viral contamination. After freeze-drying, the formulation maintains a well-defined, fluffy, pancake-like shape and exhibits low residual moisture. Furthermore, the formulation provides excellent vaccine protection without the addition of human albumin. DETAILED DESCRIPTION

[0034] The following examples further illustrate the content of this application, but should not be construed as limiting the present application. Without departing from the spirit and substance of this application, modifications or substitutions made to the methods, steps or conditions of this application are within the scope of this application.

[0035] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0036] The following preparation examples 1-24, embodiments 1-49, comparative examples 1-2 and performance testing tests further illustrate the present application in detail.

[0037] Preparation Example

[0038] Preparation Example 1-24

[0039] Preparation Example 1-24 provides a lyoprotectant.

[0040] The difference between the above preparation examples is that the addition amount of each component in the lyoprotectant is shown in Table 1.

[0041] According to the addition amount of each component in Table 1, arginine, sodium glutamate, isoleucine, glycine, trehalose, diethylaminoethyl dextran, and polyvinyl pyrrolidone were mixed uniformly and dissolved in a PBS solution to obtain a PBS mixed solution; the above PBS mixed solution was then sterilized and filtered using a 0.22 μm microporous filter membrane to obtain a lyoprotectant.

[0042] Table 1 Addition amount of each component in the freeze-dried protective agent provided in Preparation Examples 1-24

[0043]

[0044]

[0045] Example

[0046] Examples 1-24

[0047] Examples 1-24 each provide a serum-free freeze-dried human rabies vaccine preparation.

[0048] The difference between the above examples is that the lyoprotectants in the serum-free lyophilized human rabies vaccine preparations and the lyoprotectants provided in Preparation Examples 1-24 were applied to Examples 1-24, respectively, as shown in Table 2.

[0049] The preparation method of the serum-free freeze-dried human rabies vaccine preparation is as follows:

[0050] 1. After the serum-free inactivated rabies vaccine strain is cultured on microcarriers at 37°C for 20 days, the virus liquid is harvested; the virus liquid is concentrated and inactivated, and finally purified by molecular sieve to obtain the serum-free rabies vaccine.

[0051] 2. Take 20 mL of serum-free rabies vaccine and 80 mL of freeze-dried protective agent, mix them thoroughly at 2-8°C, bottle and subpackage to obtain the vaccine semi-finished product.

[0052] 3. Place the packaged vaccine in a freeze dryer and pre-freeze it at -45°C. Maintain the temperature for 3 hours after reaching the lowest temperature. Then, vacuum the vaccine to a pressure of 0.003 mbar.

[0053] 4. Enter the sublimation drying stage, raise the temperature to -29 ° C, and the time to reach the final temperature is 7 hours, and maintain the final temperature for 6 hours;

[0054] 5. Enter the desorption and drying stage, increase the temperature to 30°C, and take 5 hours to reach the final temperature. After reaching the final temperature, maintain it for 4 hours to obtain the serum-free freeze-dried human rabies vaccine preparation.

[0055] Table 2 Lyoprotectants in serum-free lyophilized human rabies vaccine preparations provided in Examples 1-24

[0056] Example Source of lyoprotectant Example Freeze-dried protection and source 1 Preparation Example 1 13 Preparation Example 13 2 Preparation Example 2 14 Preparation Example 14 3 Preparation Example 3 15 Preparation Example 15 4 Preparation Example 4 16 Preparation Example 16 5 Preparation Example 5 17 Preparation Example 17 6 Preparation Example 6 18 Preparation Example 18 7 Preparation Example 7 19 Preparation Example 19 8 Preparation Example 8 20 Preparation Example 20 9 Preparation Example 9 21 Preparation Example 21 10 Preparation Example 10 22 Preparation Example 22 11 Preparation Example 11 23 Preparation Example 23 12 Preparation Example 12 24 Preparation Example 24

[0057] Examples 25-27

[0058] Examples 25-27 each provide a serum-free freeze-dried human rabies vaccine preparation.

[0059] The difference between the above embodiment and embodiment 2 is that the weight ratio of the serum-free rabies vaccine to the lyoprotectant in the serum-free lyophilized human rabies vaccine preparation is as shown in Table 3.

[0060] The preparation methods of the serum-free freeze-dried human rabies vaccine preparations provided in Examples 25-27 are as follows:

[0061] (1) After the serum-free inactivated rabies vaccine strain is cultured on microcarriers at 37°C for 20 days, the virus liquid is harvested; the virus liquid is concentrated and inactivated, and finally purified by molecular sieve to obtain the serum-free rabies vaccine.

[0062] (2) Take 20 mL of serum-free rabies vaccine and 80 mL of freeze-dried protective agent, mix them thoroughly at 2-8°C, bottle them, and then package them to obtain a semi-finished vaccine product.

[0063] (3) Place the packaged vaccine in a freeze dryer and pre-freeze it at -45°C. Maintain the temperature for 3 hours after reaching the lowest temperature. Then, vacuum the vaccine to a pressure of 0.003 mbar.

[0064] (4) Entering the sublimation drying stage, the temperature is raised to -29 °C, the time to reach the final temperature is 7 h, and the final temperature is maintained for 6 h;

[0065] (5) Entering the desorption and drying stage, the temperature is raised to 30°C, and the time to reach the final temperature is 5 hours. After reaching the final temperature, it is maintained for 4 hours to obtain a serum-free freeze-dried human rabies vaccine preparation.

[0066] Table 3 Weight ratio of serum-free rabies vaccine and lyophilized protective agent in Examples 25-27

[0067]

[0068] Examples 28-49

[0069] Examples 28-49 each provide a serum-free freeze-dried human rabies vaccine preparation.

[0070] The differences between the above embodiment and embodiment 2 are: in the preparation process of the serum-free freeze-dried human rabies vaccine preparation, the lowest temperature in the pre-freezing stage, the holding time after reaching the lowest temperature; the vacuum pressure; the final temperature in the sublimation drying stage, the time to reach the final temperature, and the holding time after reaching the final temperature; the final temperature in the desorption drying stage, the time to reach the final temperature, and the holding time after reaching the final temperature.

[0071] Table 4 Temperature, pressure and time during the preparation of serum-free freeze-dried human rabies vaccine formulations in Examples 28-49

[0072]

[0073]

[0074] Comparative Example

[0075] Comparative Examples 1-3

[0076] Comparative Examples 1-3 respectively provide a serum-free freeze-dried human rabies vaccine preparation.

[0077] Comparative Example 1

[0078] The difference between Comparative Example 1 and Example 2 is that no diethylaminoethyl dextran is added to the serum-free freeze-dried human rabies vaccine freeze-drying protectant.

[0079] Comparative Example 2

[0080] The difference between Comparative Example 2 and Example 2 is that polyvinyl pyrrolidone is not added to the lyoprotectant of the serum-free lyophilized human rabies vaccine.

[0081] Comparative Example 3

[0082] The difference between Comparative Example 3 and Example 2 is that diethylaminoethyl dextran and polyvinyl pyrrolidone are not added to the serum-free freeze-dried human rabies vaccine freeze-drying protectant.

[0083] The preparation method of the serum-free freeze-dried human rabies vaccine preparation provided in Comparative Examples 1-3 is as follows:

[0084] (1) After the serum-free inactivated rabies vaccine strain is cultured on microcarriers at 37°C for 20 days, the virus liquid is harvested; the virus liquid is concentrated and inactivated, and finally purified by molecular sieve to obtain the serum-free rabies vaccine.

[0085] (2) Take 20 mL of serum-free rabies vaccine and 80 mL of freeze-dried protective agent, mix them thoroughly at 2-8°C, bottle them, and then package them to obtain a semi-finished vaccine product.

[0086] (3) Place the packaged vaccine in a freeze dryer and pre-freeze it at -45°C. Maintain the temperature for 3 hours after reaching the lowest temperature. Then, vacuum the vaccine to a pressure of 0.003 mbar.

[0087] (4) Entering the sublimation drying stage, the temperature is raised to -29 °C, the time to reach the final temperature is 7 h, and the final temperature is maintained for 6 h;

[0088] (5) Entering the desorption and drying stage, the temperature is raised to 30°C, and the time to reach the final temperature is 5 hours. After reaching the final temperature, it is maintained for 4 hours to obtain a serum-free freeze-dried human rabies vaccine preparation.

[0089] Performance testing

[0090] The freeze-drying effect, residual moisture and potency of the serum-free freeze-dried human rabies vaccine preparations provided in Examples 1-49 and Comparative Examples 1-3 were tested, and an accelerated thermal stability test was performed. The test results are shown in Table 5.

[0091] The residual moisture content shall be determined in accordance with Part III (Appendix VIID) of the Pharmacopoeia of the People's Republic of China and shall not exceed 3.0%.

[0092] The potency was determined according to the method of Part III (General Rule 3503) of the Pharmacopoeia of the People's Republic of China.

[0093] Accelerated thermal stability test: The serum-free freeze-dried human rabies vaccine preparation was stored at 4°C and 37°C for 28 days, respectively. The potency of the serum-free freeze-dried human rabies vaccine preparation was then tested after 0 days and 28 days of storage. The potency decline rate of the serum-free freeze-dried human rabies vaccine stored at 37°C was calculated according to the formula.

[0094] Titer reduction rate (%) = (titer at 37°C on day 0 - titer at 37°C on day 28) / titer at 37°C on day 0

[0095] Table 5 Test results of the freeze-dried vaccine preparations provided in Examples 1-49 and Comparative Examples 1-3

[0096]

[0097]

[0098]

[0099] From the above data, it can be seen that Example 2 has a good appearance, Example 2 has the lowest residual moisture compared with Example 1, Examples 3-49, and Comparative Examples 1-3, the protective effect of the vaccine of Example 2 is good, and Example 2 has the lowest potency decrease rate compared with Example 1, Examples 3-49, and Comparative Examples 1-3.

[0100] By comparing the test results of Example 2 with those of Comparative Examples 1-3, it can be seen that when diethylaminoethyl dextran is not added to the lyophilized protective agent of the serum-free freeze-dried human rabies vaccine, the protective effect of the protective agent on the vaccine is significantly reduced; when polyvinylpyrrolidone is not added to the lyophilized protective agent of the serum-free freeze-dried human rabies vaccine, the protective effect of the protective agent on the vaccine is significantly reduced, and the residual moisture content is significantly increased.

[0101] In summary, the serum-free freeze-dried human rabies vaccine lyoprotectant formulation provided herein contains no human or animal-derived ingredients, eliminating the potential risk of exogenous viral contamination. Furthermore, the protectant provides excellent protection for the vaccine. Furthermore, the lyophilized formulation has low residual moisture, improving the vaccine's stability during storage.

[0102] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A serum-free freeze-dried human rabies vaccine freeze-drying protectant, characterized in that: The freeze-drying protective agent comprises the following raw materials in parts by weight: 1-10 parts of arginine, 10-20 parts of sodium glutamate, 1-10 parts of isoleucine, 0.5-15 parts of glycine, 50-80 parts of trehalose, 10-50 parts of diethylaminoethyl dextran, and 20-60 parts of polyvinyl pyrrolidone.

2. The serum-free freeze-dried human rabies vaccine lyoprotectant according to claim 1, characterized in that The freeze-drying protective agent comprises the following raw materials in parts by weight: 1-7 parts of arginine, 10-16 parts of sodium glutamate, 5-10 parts of isoleucine, 0.5-10 parts of glycine, 50-80 parts of trehalose, 10-40 parts of diethylaminoethyl dextran, and 20-50 parts of polyvinyl pyrrolidone.

3. The serum-free freeze-dried human rabies vaccine lyoprotectant according to claim 1, characterized in that The freeze-drying protective agent comprises the following raw materials in parts by weight: 5 parts of arginine, 12 parts of sodium glutamate, 6 parts of isoleucine, 10 parts of glycine, 60 parts of trehalose, 20 parts of diethylaminoethyl dextran, and 40 parts of polyvinyl pyrrolidone.

4. The method for preparing the serum-free freeze-dried human rabies vaccine lyoprotectant according to any one of claims 1 to 3, characterized in that: After dissolving each component in PBS solution or water for injection, sterilize and filter using a microporous filter membrane to obtain the product.

5. A freeze-dried vaccine preparation, characterized in that: The vaccine is prepared by mixing the serum-free freeze-dried human rabies vaccine freeze-dried protective agent according to any one of claims 1 to 3 with the vaccine.

6. The method for preparing the freeze-dried vaccine preparation according to claim 5, characterized in that: The following steps are involved: (1) Mixing the vaccine with a lyophilized protective agent to obtain a semi-finished product solution; (2) The semi-finished product solution is pre-frozen, vacuumed, sublimated and dried, and desorbed to obtain a freeze-dried vaccine preparation.

7. The method for preparing the freeze-dried vaccine preparation according to claim 6, characterized in that: The weight ratio of the vaccine to the lyophilized protective agent is 1:(3-6).

8. The method for preparing the freeze-dried vaccine preparation according to claim 6, characterized in that: The minimum temperature in the pre-freezing stage is -45-50°C, and the temperature is maintained for 2-4 hours after reaching the minimum temperature; the vacuum pressure is 0.001-0.003 mbar.

9. The method for preparing the freeze-dried vaccine preparation according to claim 6, characterized in that: The final temperature of the sublimation drying stage is -25°C to -30°C, the time to reach the final temperature is 6-9 hours, and the final temperature is maintained for 5-7 hours after reaching the final temperature.

10. The method for preparing the freeze-dried vaccine preparation according to claim 6, characterized in that: The final temperature of the desorption drying stage is 25° C. to 30° C., the time to reach the final temperature is 3-5 hours, and the final temperature is maintained for 2-4 hours after reaching the final temperature.

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