Freeze-drying method suitable for large-scale preparation of hepatitis A attenuated live vaccine

Abstract

The invention relates to the technical field of vaccine preparation, in particular to a freeze-drying method suitable for large-scale preparation of hepatitis A attenuated live vaccines. The invention relates to a freeze-drying method suitable for large-scale preparation of a hepatitis A attenuated live vaccine, which comprises the following steps: loading a semi-finished product of the hepatitis A attenuated live vaccine to be freeze-dried into a freeze-drying box, and then executing a freeze-drying procedure of two-stage drying. According to the invention, a multi-step freeze-drying path with multi-temperature gradient combination and multi-vacuum degree gradient is arranged on the large-scale freeze dryer to realize the microscopic change of the vaccine in the freeze-drying process, so that the efficient conversion from a laboratory to large-scale production is realized, the total freeze-drying time is less than 37 hours, the total time is shortened by about 50%, the production efficiency is favorably improved, and energy conservation and consumption reduction are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, in particular to a freeze-drying method suitable for large-scale preparation of attenuated live hepatitis A vaccine. Background Art

[0002] The freeze-drying method for the live attenuated hepatitis A vaccine primarily includes the following steps: 1. Virus culture; 2. Preparation of a protective agent; 3. Prefreezing; 4. Primary drying; 5. Secondary drying; 6. Packaging and storage. These steps enable the preparation of a stable live attenuated hepatitis A vaccine. Small-scale trials of freeze-dried dosage forms in an experimental freeze-dryer yield good quality freeze-dried products. However, large-scale production, when applying the laboratory process to a large-scale production freeze-dryer, presents the following challenges: 1. Significant loss of vaccine virus activity occurs during the freeze-drying process, significantly reducing viral infectivity titers after freeze-drying; 2. The freeze-dried cakes of some samples are smaller than the frozen volume, collapse in shape, or have bottom defects, significantly impacting both moisture content and activity; 3. The production cycle is excessively long, resulting in low efficiency and high energy consumption.

[0003] The freeze-drying process results in a significant loss of infectivity titer because the survival of viral infectivity is closely linked to the thermodynamic processes and pathways experienced by the vaccine, both during production and storage and transportation. Vaccine activity (viral infectivity) depends on specific thermodynamic directions and limits. Directions that reduce the surface free energy of viral particles maintain viral infectivity. Conversely, directions that increase the surface free energy of viral particles beyond a certain limit lead to a loss of infectivity. This criterion guides the setting of state parameters for each step of the freeze-drying process, effectively improving the scientific nature of freeze-drying process design and the likelihood of maintaining viral infectivity.

[0004] Therefore, there is an urgent need to innovate and optimize laboratory processes in order to establish a freeze-drying process with an almost intact infectious titer, a loose and plump appearance, and a moisture content of less than 3%, which is suitable for fully automatic programmed operation in a large-scale production freeze dryer. Summary of the Invention

[0005] The present invention relates to a large-scale freeze-drying production process for a live attenuated viral vaccine, and is particularly suitable for a freeze-drying process for large-scale preparation of freeze-dried live attenuated hepatitis A vaccines. The process can effectively reduce the "passed" loss of infectious titer during the large-scale freeze-drying production of live attenuated hepatitis A vaccines, thereby improving vaccine stability, shortening the production cycle, and promoting energy conservation and consumption reduction.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a freeze-drying method suitable for large-scale preparation of a live attenuated hepatitis A vaccine. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, a stock solution of the live attenuated hepatitis A vaccine virus is mixed with a freeze-drying protective agent. The resulting semi-finished product is packaged into 2R-type controlled injection vials using a fully automatic filling line. The vials filled with the vaccine are sequentially transferred to each plate layer in a freeze-drying chamber in a layered, fully loaded manner. The freeze-drying chamber door is closed, and the freeze-drying procedure described in the following table (Table 1) is performed: Table 1 .

[0007] Preferably, after completing the secondary drying procedure, the process further includes step 29: pressure rise cycle detection, step 30: vacuum plugging, and step 31: freeze drying chamber partition refrigeration.

[0008] Preferably, the method for the pressure rise cycle detection is: close the septum valve for 4 to 6 minutes. If the vacuum rebound is less than 30 μbar, the moisture content of the finished vaccine product is determined to be qualified, and the next sequence is entered; otherwise, open the septum valve, extend the set time of step 28 by 28 to 32 minutes, close the septum valve for 4 to 6 minutes, detect the vacuum rebound value, if it is less than 30 μbar, enter the next sequence, otherwise, repeat the detection operation of this step until it is qualified, and the detection can be repeated up to 10 times.

[0009] Preferably, the vacuum plugging method is: automatic plugging twice under extreme vacuum, maintaining the plugging for 55 to 65 seconds each time.

[0010] Preferably, the method for refrigerating the freeze-drying box partition is: setting the temperature to 14-16° C., the air pressure to 0.8-1.0 atm, and waiting for unpacking.

[0011] Preferably, the semi-finished attenuated live hepatitis A vaccine to be freeze-dried is packaged in vials at a volume of 0.5-1.0 ml / bottle; the specification of the vials is 2R.

[0012] Preferably, the semi-finished attenuated live hepatitis A vaccine to be freeze-dried is prepared by mixing a stock solution of attenuated live hepatitis A vaccine virus and a freeze-drying protective agent in a volume ratio of 1-2:1-2.

[0013] Preferably, the infectivity titer of the attenuated live hepatitis A vaccine virus stock solution is 7-8 lgCCID 50 / ml.

[0014] Preferably, the moisture content of the freeze-dried attenuated live hepatitis A vaccine product is less than 3.0%.

[0015] Preferably, the lyoprotectant uses water as solvent and comprises the following components in the following mass concentrations: trehalose 4-8 g / 100 ml and glycine 8-12 g / 100 ml.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Prevent bottom defects: Figure 7 The frozen state of the vaccine in the vial is depicted in this figure. The sublimation process from top to bottom is divided into linear and nonlinear regions (D represents a plane). Segment AB is the cylindrical linear region, with a uniform cross-section from top to bottom. Segment BC is the nonlinear region, with a gradually decreasing cross-section from top to bottom. Traditional freeze-drying processes use the same constant temperature and pressure sublimation process for both segments AB and BC, potentially leading to bottom shrinkage and collapse in the BC segment. This invention utilizes a cooling sublimation process for the BC segment, eliminating this bottom shrinkage risk.

[0017] 2. This invention utilizes a multi-step freeze-drying process incorporating multiple temperature gradients and vacuum gradients in a large freeze-dryer to achieve microscopic changes in the vaccine during the freeze-drying process, thereby enabling efficient transition from laboratory to large-scale production. The total freeze-drying time is less than 37 hours, a reduction of approximately 50%. This helps improve production efficiency and contributes to energy conservation and consumption reduction.

[0018] 3. The present invention uses the research on "Thermodynamic Directions and Limits of Preservation of Infectious Titer of Live Attenuated Viral Vaccines" as a criterion to guide the setting of state parameters for each step of the freeze-drying process, effectively improving the scientific nature of the freeze-drying process setting and the possibility of intact viral infectivity.

[0019] The present invention divides the sublimation process from top to bottom into linear and nonlinear regions and implements drying operations separately, avoiding the traditional freeze-drying process that sets the sublimation process to a constant temperature and constant pressure operation throughout the process, which causes the bottom of the vaccine to shrink and collapse. By setting a multi-step freeze-drying path with multiple temperature gradients and multiple vacuum gradients on a large freeze dryer, the microscopic changes of the vaccine during the freeze-drying process are achieved, thereby achieving efficient conversion from laboratory to large-scale production. The total freeze-drying time of the freeze-drying process of the present invention is less than 37 hours, which is about 50% shorter than the total time of the prior art, which helps to improve production efficiency and achieve the sustainable development goals of energy conservation, consumption reduction, pollution reduction, efficiency improvement, etc. It is suitable as a freeze-drying process for converting laboratory results into large-scale production of viral attenuated live vaccines and active extract preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0021] Figure 1 This is the freeze-drying curve of Experimental Example 1.

[0022] Figure 2 This is the freeze-drying curve of Experimental Example 2.

[0023] Figure 3 This is the freeze-drying curve of Experimental Example 3.

[0024] Figure 4 This is the freeze-drying curve of Experimental Example 4.

[0025] Figure 5 This is the freeze-drying curve of Experimental Example 5.

[0026] Figure 6 This is the freeze-drying curve of Experimental Example 6.

[0027] Figure 7 This is a diagram of the structure of a syringe bottle. DETAILED DESCRIPTION

[0028] The present invention provides a freeze-drying method suitable for large-scale preparation of a live attenuated hepatitis A vaccine. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, a stock solution of the live attenuated hepatitis A vaccine virus is mixed with a freeze-drying protective agent. The resulting semi-finished product is packaged into 2R-type controlled injection vials using a fully automatic filling line. The vials filled with the vaccine are sequentially transferred to each plate layer in a freeze-drying chamber in a layered and fully loaded manner. The freeze-drying chamber door is closed, and the freeze-drying procedure in Table 1 is executed.

[0029] Preferably, the freeze-drying procedure in the following table (Table 2) is performed: Table 2 .

[0030] In the present invention, after the secondary drying process is completed, the steps further include step 29: pressure rise cycle detection, step 30: vacuum plugging, and step 31: freeze drying chamber partition refrigeration.

[0031] In the present invention, the method of pressure rise cycle detection is: close the septum valve for 4 to 6 minutes. If the vacuum rebound is less than 30 μbar, the moisture content of the finished vaccine product is determined to be qualified, and the next sequence is entered; otherwise, open the septum valve, extend the setting time of step 28 by 28 to 32 minutes, close the septum valve for 4 to 6 minutes, detect the vacuum rebound value, if it is less than 30 μbar, enter the next sequence, otherwise, repeat the detection operation of this step until it is qualified, and the detection can be repeated up to 10 times; preferably, close the septum valve for 5 minutes. If the vacuum rebound is less than 30 μbar, the moisture content of the finished vaccine product is determined to be qualified, and the next sequence is entered; otherwise, open the septum valve, extend the setting time of step 28 by 30 minutes, close the septum valve for 5 minutes, detect the vacuum rebound value, if it is less than 30 μbar, enter the next sequence, otherwise, repeat the detection operation of this step until it is qualified, and the detection can be repeated up to 10 times.

[0032] In the present invention, the vacuum plugging method is: automatic plugging twice under ultimate vacuum, each time maintaining the plugging for 55 to 65 seconds; preferably 57 to 63 seconds; more preferably 60 seconds.

[0033] In the present invention, the method for refrigerating the freeze drying chamber partition is: setting the temperature to 14-16°C, the air pressure to 0.8-1.0atm, and waiting for unloading; preferably, setting the temperature to 15°C and the air pressure to 1atm.

[0034] In the present invention, the semi-finished attenuated live hepatitis A vaccine to be freeze-dried is packaged in vials at a volume of 0.5-1.0 ml per vial, preferably 0.5 ml.

[0035] In the present invention, the specification of the vial is 2R.

[0036] In the present invention, the semi-finished attenuated live hepatitis A vaccine to be freeze-dried is prepared by mixing the attenuated live hepatitis A vaccine virus stock solution and the freeze-drying protective agent in a volume ratio of 1-2:1-2; preferably 1:1.

[0037] In the present invention, the infectivity titer of the attenuated live hepatitis A vaccine virus stock solution is 7-8 lgCCID 50 / ml; preferably 7.51gCCID 50 / ml.

[0038] In the present invention, the water content of the freeze-dried attenuated live hepatitis A vaccine product is less than 3.0%.

[0039] In the present invention, the type of the lyoprotectant is the lyoprotectant, which uses water as a solvent and includes the following components in mass concentrations: trehalose 4-8g / 100ml, glycine 8-12g / 100ml; preferably trehalose 6g / 100ml, glycine 10g / 100ml.

[0040] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] In the following examples, the lyoprotectant uses water as solvent and is composed of the following components in the following mass concentrations: trehalose 6 g / 100 ml and glycine 10 g / 100 ml.

[0042] Example 1

[0043] A freeze-drying process for large-scale preparation of freeze-dried live attenuated hepatitis A vaccine is provided, wherein the freeze-dried live attenuated hepatitis A vaccine is prepared through a multi-step freeze-drying process combining multiple temperature gradients with multiple vacuum gradients as follows: (a) In accordance with the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, the attenuated live hepatitis A vaccine virus stock solution and the lyophilization protective agent were mixed in a 1:1 volume ratio. The resulting semi-finished product was dispensed into 2R-type controlled injection vials using a fully automated filling line at a rate of 0.5 ml per vial (one dose per vial); (b) Transfer the filled vaccine vials to the various layers of the freeze-drying chamber in a layered, fully loaded manner. Close the freeze-drying chamber door and execute the freeze-drying program in Table 2: Step 29. Pressure rise cycle test: Close the septum valve for 5 minutes. If the vacuum rebound is less than 30μbar, the vaccine moisture content is determined to be qualified and go to step 30; otherwise, open the septum valve and continue step 28 for 30 minutes. Close the septum valve for 5 minutes and test the vacuum rebound value. If it is less than 30μbar, go to step 30. Otherwise, repeat this step test operation until it is qualified. The test can be repeated up to 10 times.

[0044] Step 30. Vacuum plugging: Automatically plug twice under extreme vacuum, maintaining plugging for 60 seconds each time.

[0045] Step 31. Freeze-drying chamber partition cooling: 15.0℃, 1atm, waiting for unpacking and capping.

[0046] Experimental Example 1: Large-scale preparation of freeze-dried live attenuated hepatitis A vaccine 1. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, the attenuated live hepatitis A vaccine virus stock solution and the lyophilization protective agent are mixed in a volume ratio of 1:1. The resulting semi-finished product is dispensed into 2R-type controlled injection vials using a fully automatic filling line at a volume of 0.5 ml per vial (one dose per vial); 2. Transfer the filled vaccine vials to the various layers of the freeze-drying chamber in a layered and fully loaded manner. Close the freeze-drying chamber door and perform the freeze-drying method of Example 1: 3. Freeze-drying curve of Experimental Example 1 Figure 1 shown.

[0047] 4. Test results of the finished product after freeze-drying: Infectious titer before freeze-drying 7.5lgCCID 50 / ml, the infectious titer of the lyophilized product was 7.0 lgCCID 50 / ml, the infectious titer of the finished product after heat stabilization was 6.67 lgCCID 50 / ml, the virus titer before and after thermal stabilization decreased by no more than 0.50 lgCCID 50 / ml; the moisture content of the finished product after freeze-drying is 1.1% (W / V%), the appearance is loose, plump and without defects, and the solution after dissolution is a colorless, transparent, clear liquid without foreign matter.

[0048] Experimental Example 2: Large-scale preparation of freeze-dried live attenuated hepatitis A vaccine 1. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, the attenuated live hepatitis A vaccine virus stock solution and the lyophilization protective agent are mixed in a volume ratio of 1:1. The resulting semi-finished product is dispensed into 2R-type controlled injection vials using a fully automatic filling line at a volume of 0.5 ml per vial (one dose per vial); 2. Transfer the filled vaccine vials to the various layers of the freeze-drying chamber in a layered and fully loaded manner. Close the freeze-drying chamber door and perform the freeze-drying method of Example 1: 3. Experimental Example 2 Freeze-drying curve Figure 2 shown.

[0049] 4. Test results of the finished product after freeze-drying: Infectious titer before freeze-drying 7.5 lgCCID 50 / ml, the infectious titer of the freeze-dried finished product is 6.67 lgCCID 50 / ml, the infectious titer of the finished product after heat stabilization was 6.67 lgCCID 50 / ml, the virus titer before and after thermal stabilization decreased by no more than 0.50 lgCCID 50 / ml; the moisture content of the finished product after freeze-drying is 1.1% (W / V%), the appearance is loose, plump and without defects, and the solution after dissolution is a colorless, transparent, clear liquid without foreign matter.

[0050] Experimental Example 3: Large-scale preparation of freeze-dried live attenuated hepatitis A vaccine 1. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, the attenuated live hepatitis A vaccine virus stock solution and the lyophilization protective agent are mixed in a volume ratio of 1:1. The resulting semi-finished product is dispensed into 2R-type controlled injection vials using a fully automatic filling line at a volume of 0.5 ml per vial (one dose per vial); 2. Transfer the filled vaccine vials to the various layers of the freeze-drying chamber in a layered and fully loaded manner. Close the freeze-drying chamber door and perform the freeze-drying method of Example 1: 3. Experimental Example 3 Freeze-drying curve Figure 3 shown.

[0051] 4. Test results of the finished product after freeze-drying: Infectious titer before freeze-drying 7.5 lgCCID 50 / ml, the infectious titer of the freeze-dried finished product was 7.17 lgCCID 50 / ml, the infectious titer of the finished product after heat stabilization was 6.67 lgCCID 50 / ml, the virus titer before and after thermal stabilization decreased by no more than 0.50 lgCCID 50 / ml; the moisture content of the finished product after freeze-drying is 1.7% (W / V%), the appearance is loose, plump and without defects, and the solution after dissolution is a colorless, transparent, clear liquid without foreign matter.

[0052] Experimental Example 4: Large-scale preparation of freeze-dried live attenuated hepatitis A vaccine 1. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, the attenuated live hepatitis A vaccine virus stock solution and the lyophilization protective agent are mixed in a volume ratio of 1:1. The resulting semi-finished product is dispensed into 2R-type controlled injection vials using a fully automatic filling line at a volume of 0.5 ml per vial (one dose per vial); 2. Transfer the filled vaccine vials to the various layers of the freeze-drying chamber in a layered and fully loaded manner. Close the freeze-drying chamber door and perform the freeze-drying method of Example 1: 3. Experimental Example 4 Freeze-drying curve Figure 4 shown.

[0053] 4. Test results of the finished product after freeze-drying: Infectious titer before freeze-drying 7.5 lgCCID 50 / ml, the infectious titer of the lyophilized product was 7.0 lgCCID 50 / ml, the infectious titer of the finished product after heat stabilization was 6.77 lgCCID 50 / ml, the virus titer before and after thermal stabilization decreased by no more than 0.50 lgCCID 50 / ml; the moisture content of the finished product after freeze-drying is 1.5% (W / V%), the appearance is loose, plump and without defects, and the solution after dissolution is a colorless, transparent, clear liquid without foreign matter.

[0054] Experimental Example 5: Large-scale preparation of freeze-dried live attenuated hepatitis A vaccine 1. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, the attenuated live hepatitis A vaccine virus stock solution and the lyophilization protective agent are mixed in a volume ratio of 1:1. The resulting semi-finished product is dispensed into 2R-type controlled injection vials using a fully automatic filling line at a volume of 0.5 ml per vial (one dose per vial); 2. Transfer the filled vaccine vials to the various layers of the freeze-drying chamber in a layered and fully loaded manner. Close the freeze-drying chamber door and perform the freeze-drying method of Example 1: 3. Experimental Example 5 Freeze-drying curve Figure 5 shown.

[0055] 4. Test results of the finished product after freeze-drying: Infectious titer before freeze-drying 7.67 lgCCID 50 / ml, the infectious titer of the lyophilized product was 7.0 lgCCID 50 / ml, the infectious titer of the finished product after heat stabilization was 6.67 lgCCID 50 / ml, the virus titer before and after thermal stabilization decreased by no more than 0.50 lgCCID 50 / ml; the moisture content of the finished product after freeze-drying is 1.3% (W / V%), the appearance is loose, plump and without defects, and the solution after dissolution is a colorless, transparent, clear liquid without foreign matter.

[0056] Experimental Example 6: Large-scale preparation of freeze-dried live attenuated hepatitis A vaccine 1. According to the requirements of the General Rules for Biological Products in Part III of the Chinese Pharmacopoeia, the attenuated live hepatitis A vaccine virus stock solution and the lyophilization protective agent are mixed in a volume ratio of 1:1. The resulting semi-finished product is dispensed into 2R-type controlled injection vials using a fully automatic filling line at a volume of 0.5 ml per vial (one dose per vial); 2. Transfer the filled vaccine vials to the various layers of the freeze-drying chamber in a layered and fully loaded manner. Close the freeze-drying chamber door and perform the freeze-drying method of Example 1: 3. Freeze-drying curve of Experimental Example 6 Figure 6 shown.

[0057] 4. Test results of the finished product after freeze-drying: Infectious titer before freeze-drying 7.33 lgCCID 50 / ml, the infectious titer of the freeze-dried finished product is 6.67 lgCCID 50 / ml, the infectious titer of the finished product after heat stabilization is 6.5logCCID 50 / ml, the virus titer before and after thermal stabilization decreased by no more than 0.50 lgCCID 50 / ml; the moisture content of the finished product after freeze-drying is 2.3% (W / V%), the appearance is loose, plump and without defects, and the solution after dissolution is a colorless, transparent, clear liquid without foreign matter.

[0058] Experimental Example 7

[0059] The relevant indicators of the freeze-dried products obtained in Experimental Examples 1 to 6 were measured, and the results are shown in Table 3. The results show that the test results of each experimental example meet the requirements of the Chinese Pharmacopoeia.

[0060] Table 3

[0061] in conclusion: ⑴ The infectivity titer of the finished virus after freeze-drying is ≥6.5 lgCCID 50 / ml; ⑵ The finished product after freeze-drying is subjected to a thermal stability test at 37°C for 72 hours, and the viral infectivity titer is ≥6.5 lgCCID 50 / ml, the virus titer decreased by no more than 0.50 lgCCID 50 / ml; ⑶ The moisture content of the finished product after freeze-drying is less than 3.0%; (4) The appearance of the finished product after freeze-drying is loose and full, without shrinkage or collapse; ⑸Effectively improve freeze-drying production efficiency: total time is less than 37 hours, which is about 50% shorter than the laboratory process.

[0062] Experimental Example 8 Long-term stability inspection cycle: 1. Shelf Life: According to the Chinese Pharmacopoeia and manufacturing specifications, the shelf life of freeze-dried live attenuated hepatitis A vaccine is 18 months (from the date of production) when stored at 2-8°C in the dark. This shelf life is verified through long-term stability studies, with regular testing of indicators such as viral titer.

[0063] 2. Long-term stability study: The entire validity period must be covered, with regular sampling and testing (0, 3, 6, 9, 12, 18, and 24 months) to assess the viral titer (no less than 6.50 lg CCID 50 / dose), moisture (≤3%), sterility and other indicators.

[0064] Table 4 Stability test titer values ​​of freeze-dried attenuated live hepatitis A vaccine (1gCCID 50 / dose)

[0065] Table 5 Moisture content of freeze-dried attenuated hepatitis A vaccine (w / v% / dose)

[0066] Table 6 Stability test of freeze-dried attenuated hepatitis A live vaccine Sterility test

[0067] As shown in Tables 4 to 6, the freeze-dried live attenuated hepatitis A vaccine provided by the present invention has good stability and remains stable after 24 months of storage. The titer value of the finished product does not change significantly, and the moisture content and sterility tests of the finished product are also qualified.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A freeze-drying method suitable for large-scale preparation of live attenuated hepatitis A vaccine, characterized in that: After the freeze-dried attenuated live hepatitis A vaccine semi-finished product is placed in the freeze-drying chamber, the freeze-drying procedure in Table 1 is performed: Table 1 。 2. The method according to claim 1, characterized in that After completing the secondary drying process, the process also includes step 29: pressure rise cycle detection, step 30: vacuum plugging, and step 31: freeze drying chamber partition refrigeration.

3. The method according to claim 2, characterized in that The method for the pressure rise cycle detection is: close the septum valve for 4 to 6 minutes. If the vacuum rebound is less than 30μbar, the moisture content of the finished vaccine product is determined to be qualified and enter the next sequence; otherwise, open the septum valve, extend the set time of step 28 by 28 to 32 minutes, close the septum valve for 4 to 6 minutes, detect the vacuum rebound value, if it is less than 30μbar, enter the next sequence, otherwise, repeat the detection operation of this step until it is qualified. The detection can be repeated up to 10 times.

4. The method according to claim 2, characterized in that The vacuum plugging method is: automatically plugging twice under extreme vacuum, and maintaining the plugging for 55 to 65 seconds each time.

5. The method according to claim 2, characterized in that The method for refrigerating the freeze-drying box partition is as follows: setting the temperature to 14-16° C., the air pressure to 0.8-1.0 atm, and waiting for unpacking.

6. The method according to claim 2, characterized in that The semi-finished attenuated live hepatitis A vaccine to be freeze-dried is packaged in vials at a volume of 0.5-1.0 ml per vial; the specification of the vials is 2R.

7. The method according to claim 2, characterized in that The attenuated live hepatitis A vaccine semi-finished product to be freeze-dried is prepared by mixing the attenuated live hepatitis A vaccine virus stock solution and a freeze-drying protective agent in a volume ratio of 1-2:1-2.

8. The method according to claim 7, characterized in that The infectivity titer of the hepatitis A attenuated live vaccine virus stock solution is 7-8 lgCCID 50 / ml.

9. The method according to claim 2, characterized in that The moisture content of the freeze-dried hepatitis A live attenuated vaccine product is less than 3.0%.

10. The method according to claim 2, characterized in that The freeze-drying protective agent uses water as a solvent and includes the following components in mass concentrations: trehalose 4-8g / 100ml and glycine 8-12g / 100ml.

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