Preparation method of varicella attenuated live vaccine, freeze-drying protective agent and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a preparation method of varicella attenuated live vaccine, a freeze-drying protective agent and application thereof, which comprises the steps of cell culture, virus infection, complex enzymatic dissociation, protective ultrasonic disruption, clarification and collection, etc. The complex enzymatic dissociation adopts EDTA and recombinant trypsin combination to loosen the cells at low temperature; the protective ultrasonic disruption adopts a buffer solution containing trehalose and recombinant human blood albumin to resuspend the cells and perform intermittent ultrasonic disruption. The present application further provides a freeze-drying protective agent which is composed of trehalose, sorbitol, L-arginine, L-histidine and polyvinylpyrrolidone. The virus harvesting efficiency of the method is high, and the virus activity loss is small; the freeze-drying protective agent has good safety and excellent stability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing a live attenuated varicella vaccine, a freeze-drying protectant, and its application. Background Technology

[0002] Chickenpox is an acute infectious disease caused by primary infection with the varicella-zoster virus (VZV). It mainly occurs in infancy and early childhood, and is characterized by fever and successive appearance of systemic red maculopapular rashes, vesicles, and scabs. It can be accompanied by serious complications such as pneumonia and encephalitis. Vaccination against chickenpox is currently the most effective measure to prevent chickenpox infection.

[0003] Varicella-zoster virus (VZV), also known as human herpesvirus type 3, is a double-stranded DNA virus with neurotropic and cutaneous properties; humans are its only host. VZV glycoprotein E is an essential component for viral replication and intercellular transmission, and is also a major target for virus-specific antibodies and T-cell responses, often selected as a candidate antigen for vaccines. In 1971, Japanese scholar Dr. Rikiaki Takahashi isolated the wild-type varicella-zoster virus Oka strain from the vesicle fluid of children with chickenpox. Using the Oka strain as a parent, he obtained the V-Oka attenuated strain through passage, which was then used to prepare a live attenuated vaccine.

[0004] Currently, the varicella live attenuated vaccine, prepared by culturing the varicella attenuated strain in human diploid cells, is the only means of preventing varicella infection. Existing technologies for preparing varicella live attenuated vaccines mainly include cell culture, virus infection, virus harvesting, and freeze-drying. However, existing technologies still have the following technical problems:

[0005] First, virus harvesting efficiency is low, and virus activity is greatly lost. Current technologies typically employ methods such as ultrasonic disruption, chemical reagents, or enzyme treatment for virus harvesting. Chemical reagent or enzyme treatment methods suffer from cumbersome processes, long preparation cycles, and low virus yields. While traditional ultrasonic cell disruption is simple, improper ultrasonic power settings or insufficient treatment time can lead to incomplete cell structure destruction, or excessively high ultrasonic energy can damage virus particles, making it difficult to achieve efficient cell disruption and virus release (see CN119367529A). For example, CN104258404B discloses a method of continuous flow ultrasonic disruption using 0.03%-0.05% EDTA solution to digest cells, followed by ultrasonic disruption at 30kHz at 2-8℃; CN101161286B discloses a method of ultrasonic disruption using EDTA to elute infected cells, followed by ultrasonic disruption at 30kHz and 0.1-0.2 seconds / ml. None of the above methods take into account the gentle dissociation of cells before ultrasonic disruption or the protection of the virus during ultrasonication, resulting in a significant loss of viral activity during harvesting.

[0006] Second, existing freeze-dried protectants pose safety risks. Currently, most freeze-dried protectants for varicella vaccines contain large molecular components such as gelatin, dextran, or human serum albumin. Gelatin is a major source of endotoxins in vaccines, which can cause fever in recipients. Most allergic reactions to varicella vaccines are caused by gelatin, which acts as a stabilizer in the vaccine (see CN104258404B). Dextran itself is a potent antigen that can trigger anaphylactic shock, with a mortality rate 2-4 times higher than that of penicillin (see CN104258404B). Human serum albumin, as a blood product, poses a potential risk of virus transmission (see CN101312742A). Although existing technologies have attempted to remove gelatin (such as CN101537186A and CN102657870A), they still contain dextran or human serum albumin, failing to completely resolve the safety issues. CN104258404B discloses a protective agent that does not contain gelatin, dextran, or human serum albumin, but its formulation contains urea and 199 culture medium. Its adaptability to the freeze-drying process and its long-term protective effect against the virus still need further verification.

[0007] Third, existing stabilizers struggle to achieve synergistic protection across multiple components. CN101312742A discloses a stabilizer for freeze-dried vaccines, whose core components are reducing monosaccharides (such as glucose and fructose) and acid antioxidants (such as aspartic acid, glutamic acid, and ascorbic acid), explicitly excluding the salt forms of these acid antioxidants. While this stabilizer can protect viral activity to some extent, it relies on the combination of reducing monosaccharides and acid antioxidants, and the synergistic effects of basic amino acids and other protective agents have not been studied. Furthermore, reducing monosaccharides may undergo Maillard reactions during long-term storage, affecting vaccine stability.

[0008] To address the aforementioned technical issues, there is an urgent need for a method to prepare a live attenuated varicella vaccine that features high virus harvesting efficiency, minimal loss of virus activity, a safe and allergen-free freeze-drying protectant, and excellent long-term stability. Summary of the Invention

[0009] To address the aforementioned technical problems, the present invention aims to provide a method for preparing a live attenuated varicella vaccine, a freeze-drying protectant, and its application. The preparation method can significantly improve virus harvesting efficiency and reduce impurity residues, while providing a safe, allergen-free, and highly stable freeze-drying protectant, thereby achieving efficient preparation and long-term stable preservation of the live attenuated varicella vaccine.

[0010] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a live attenuated varicella vaccine virus stock solution, comprising the following steps: (1) Cell culture: MRC-5 human diploid cells were expanded and passaged within 35 generations; (2) Viral infection: After the MRC-5 human diploid cells have grown into a uniform and dense monolayer of cells, Oka strain virus is added to infect the cells for infection and culture. (3) Compound enzyme dissociation: After the cell lesion reaches more than 70%, discard the culture medium, add the compound dissociation solution, and treat at 20-37℃ for 2-5 minutes to loosen the cells from the culture surface; (4) Protective sonication: Discard the composite dissociation solution, add buffer containing a protective agent to resuspend the cells, and perform intermittent sonication under ice bath conditions; (5) Clarification and collection: The lysate after sonication is filtered and the filtrate is collected, which is the original virus solution.

[0011] As a preferred technical solution, step (5) specifically involves filtering the lysate after sonication using a 40μm filter cartridge and collecting the filtrate, which is the original virus solution.

[0012] As a preferred technical solution, the composite dissociation solution in step (3) contains 0.03%-0.05% EDTA and 0.01%-0.02% recombinant trypsin by mass and volume, and the solvent is PBS buffer.

[0013] As a preferred technical solution, the protective agent in step (4) is trehalose with a mass-volume percentage of 2%-3% and recombinant human serum albumin with a mass-volume percentage of 0.5%-1%, and the solvent is PBS buffer.

[0014] As a preferred technical solution, the process parameters of the intermittent ultrasonic crushing in step (4) are: ultrasonic power 100 W-150 W, ultrasonic time 1-2 seconds, interval time 2-3 seconds, and total number of ultrasonic cycles 4-6.

[0015] Secondly, the present invention provides a lyophilized protectant for a live attenuated varicella vaccine, wherein each liter of the lyophilized protectant comprises the following components: Trehalose 45-55 g; Sorbitol 12-18 g; L-arginine 6-10 g; L-histidine 3-5 g; Polyvinylpyrrolidone 12-18 g; The balance is 0.01-0.05 mol / L phosphate buffer, and the pH of the lyophilization protectant is 7.2-7.4.

[0016] As a preferred technical solution, the polyvinylpyrrolidone is PVP K30.

[0017] As a preferred technical solution, the mass ratio of trehalose to sorbitol is (2.5-4.6):1.

[0018] As a preferred technical solution, the mass ratio of L-arginine to L-histidine is (1.5-2.5):1.

[0019] Thirdly, the present invention provides a method for preparing a live attenuated varicella vaccine, comprising the following steps: (1) Prepare the virus stock solution using the method provided in the first aspect above; (2) Determine the titer of the original virus solution, add the freeze-drying protectant described in the second aspect, and dilute the virus titer to the preset target value to obtain a semi-finished product; (3) After the semi-finished product is packaged and freeze-dried, the varicella attenuated live vaccine is obtained.

[0020] As a preferred technical solution, the freeze drying in step (3) includes: pre-freezing to below -45°C and holding for 2-3 hours; primary drying at 10-20 Pa pressure and maintaining a partition temperature of -15°C for 15-20 hours; secondary drying at 5 Pa pressure and maintaining a partition temperature of 25-30°C for 4-6 hours.

[0021] Fourthly, the present invention provides a live attenuated varicella vaccine, prepared by using the method described in any one of the above-mentioned methods to prepare the virus stock solution, or by using the freeze-drying protectant described in any one of the above-mentioned methods, or by using the method described in any one of the above-mentioned methods.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: First, this invention significantly improves virus harvesting efficiency and reduces virus activity loss. By employing a composite dissociation solution for gentle cell dissociation, cells are loosened from the culture surface without rupture, avoiding damage to the virus from over-digestion. This is further enhanced by intermittent ultrasonic disruption using a buffer solution containing a protective agent, effectively buffering the thermal and mechanical stresses generated by ultrasound. This process significantly increases virus titers compared to traditional direct ultrasonic methods, while drastically reducing the amount of residual host cell proteins.

[0023] Secondly, this invention provides a highly safe lyophilization protectant. This protectant uses polyvinylpyrrolidone (PVP) instead of dextran as the lyophilization backbone forming agent, avoiding the risk of anaphylactic shock that dextran may cause. Furthermore, the use of L-arginine and L-histidine in the lyophilization protectant avoids the potential risk of virus transmission associated with blood-derived human serum albumin. Vaccines prepared using this protectant fundamentally ensure the safety of vaccine administration.

[0024] Furthermore, the protective agent of this invention achieves a synergistic protective effect of multiple components, which significantly improves the stability of the vaccine. This protective agent enables the vaccine to maintain good viral activity under long-term storage and heat-accelerated destruction conditions, and its stability is significantly better than that of existing technology formulations.

[0025] In summary, this invention achieves end-to-end protection from virus preparation to finished product preservation through synergistic innovation in virus harvesting technology and freeze-drying protectants. While ensuring high virus titer, high purity, and excellent stability, it completely eliminates the risks of allergens and animal origins associated with traditional components such as gelatin, dextran, and human serum albumin, significantly reduces endotoxin content, and meets clinical reconstitution requirements. This invention represents a safe, efficient, and stable technology for preparing a live attenuated varicella vaccine. Detailed Implementation

[0026] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and are therefore only examples and should not be used to limit the scope of protection of the present invention. Example

[0027] The purpose of this embodiment is to provide a method for preparing a live attenuated varicella vaccine, including the following steps: 1. Cell Culture Human diploid cells of the MRC-5 line (derived from ATCC, CCL-171) were amplified and passaged in MEM medium (pH 7.3) containing 10% newborn calf serum at 37°C in a 5% CO2 incubator. The cell passage ratio was 1:2 to 1:4, with passages every 3-5 days until passage 30 (within 35 passages). Cells were then used after forming a uniform, dense, and complete monolayer.

[0028] 2. Viral infection After the cells grew into a uniform and dense monolayer, the medium was replaced with MEM maintenance medium at pH 7.3 containing 2% newborn calf serum (NBS). Oka strain virus (source: ATCC, VR-1832) was added at an MOI of 0.03, and the cells were cultured at 35°C and 5% CO2. Forty-eight hours after infection, the culture medium was discarded, and the cell surface was washed twice with sterile PBS buffer at pH 7.3 to thoroughly remove residual NBS. The medium was then replaced with MEM medium at pH 7.3 without NBS, and the cells were cultured at 35°C. When cytopathic effects reached 75% or higher, the virus was ready for harvesting.

[0029] 3. Dissociation of complex enzymes Discard the culture medium and add a composite dissociation solution at a volume of 150 ml per cell factory layer. This dissociation solution is PBS buffer (pH 7.3) containing 0.04% (w / v) EDTA and 0.015% (w / v) recombinant trypsin. Gently shake horizontally at 37°C for 2 minutes to completely loosen the cells from the culture surface while maintaining their morphology and preventing cell detachment and disruption. Discard the composite dissociation solution immediately after treatment.

[0030] 4. Protective ultrasonic fragmentation After discarding the recombinant dissociation solution, the cells were immediately resuspended in PBS buffer containing 2.5% (w / v) trehalose and 0.8% (w / v) recombinant human serum albumin, at a volume of 100 ml / layer of cell factory. The cell suspension was transferred to a pre-cooled sonication cup and subjected to intermittent sonication disruption under ice bath conditions throughout the process. The core sonication parameters were: sonication power 120 W, sonication frequency 20 kHz, sonication time 1.5 seconds, interval time 2.5 seconds, and a total of 5 sonication cycles.

[0031] 5. Clarification and Collection The cell lysate after sonication was filtered through a filter cartridge with a pore size of 40 μm, and the filtrate was collected. The resulting filtrate was the varicella virus stock solution.

[0032] 6. Preparation of freeze-drying protectant Prepare 1 L of lyophilization protectant according to the formula in Table 1. Each liter of the lyophilization protectant consists of the following components. The entire process should be carried out under aseptic conditions: Table 1. Composition of the freeze-drying protectant in Example 1

[0033] Add the above components sequentially to the phosphate buffer solution, stir at low speed until completely dissolved, adjust the pH to 7.3 precisely with 1N hydrochloric acid or 1N sodium hydroxide, filter through a 0.22μm sterile filter membrane for sterilization, and store temporarily at 2-8℃ for later use.

[0034] 7. Semi-finished product preparation and freeze-drying Take the prepared virus stock solution, determine its virus titer, and then dilute it with lyophilization protectant according to the preset target titer for the semi-finished product (preset titer is 5.2 lgPFU / mL). After stirring evenly, the vaccine semi-finished product is obtained.

[0035] The semi-finished product was dispensed into 2ml vials, with each vial containing 0.6ml. After partially sealing the vials, they were immediately placed in a freeze dryer and subjected to vacuum freeze-drying according to the following process: Pre-freezing stage: The temperature of the partition is uniformly reduced to -45℃ at a rate of 1℃ / min, and kept at a constant temperature for 2.5 hours; First drying: Vacuum up to 15 Pa, raise the temperature of the partition to -15℃ at a rate of 0.5℃ / min, and maintain the temperature for 18 hours; Secondary drying: The vacuum level is reduced to below 3 Pa, and the temperature of the partition is increased to 28°C at a rate of 0.5°C / min and maintained at a constant temperature for 5 hours; After freeze-drying, the vaccine is fully stoppered under vacuum, and then capped after being removed from the box to obtain the finished varicella live attenuated vaccine.

[0036] Example 2 The purpose of this embodiment is to provide a method for preparing a live attenuated varicella vaccine, including the following steps: 1. Cell Culture Human diploid cells of the MRC-5 line (derived from ATCC, CCL-171) were amplified and passaged in MEM medium (pH 7.3) containing 10% newborn calf serum at 37°C in a 5% CO2 incubator. The cell passage ratio was 1:2–1:4, with passages every 3–5 days until passage 28 (within 35 passages). Cells were then used after forming a uniform, dense, and complete monolayer.

[0037] 2. Viral infection After the cells grew into a uniform and dense monolayer, the medium was replaced with MEM maintenance medium at pH 7.3 containing 2% newborn calf serum (NBS). Oka strain virus (source: ATCC, VR-1832) was added at an MOI of 0.028, and the cells were cultured at 35°C and 5% CO2. Fifty hours after infection, the culture medium was discarded, and the cell surface was washed twice with sterile PBS buffer at pH 7.3 to thoroughly remove residual NBS. The medium was then replaced with MEM medium at pH 7.3 without NBS, and the cells were cultured at 35°C. When cytopathic effects reached 75% or higher, the virus was ready for harvesting.

[0038] 3. Dissociation of complex enzymes Discard the culture medium and add a composite dissociation solution at a volume of 150 ml per cell factory layer. This dissociation solution is PBS buffer (pH 7.3) containing 0.04% (w / v) EDTA and 0.015% (w / v) recombinant trypsin. Gently shake horizontally at 20°C for 5 minutes to completely loosen the cells from the culture surface while maintaining their morphology and preventing cell detachment and disruption. Discard the composite dissociation solution immediately after treatment.

[0039] 4. Protective ultrasonic fragmentation After discarding the recombinant dissociation solution, the cells were immediately resuspended in PBS buffer containing 2.5% (w / v) trehalose and 0.8% (w / v) recombinant human serum albumin, at a volume of 100 ml / layer of cell factory. The cell suspension was transferred to a pre-cooled sonication cup and subjected to intermittent sonication disruption under ice bath conditions throughout the process. The core sonication parameters were: sonication power 120 W, sonication frequency 20 kHz, sonication time 1.5 seconds, interval time 2.5 seconds, and a total of 4 sonication cycles.

[0040] 5. Clarification and Collection Same as Example 1.

[0041] 6. Preparation of freeze-drying protectant The same lyophilization protectant formulation as in Example 1 (see Table 1) was used, namely, each liter of lyophilization protectant contained 50 g of trehalose, 15 g of sorbitol, 8 g of L-arginine, 4 g of L-histidine, and 15 g of polyvinylpyrrolidone, with the balance being 0.01-0.05 mol / L phosphate buffer at pH 7.2-7.4. The preparation of 1 L of lyophilization protectant was carried out under aseptic conditions throughout the process.

[0042] 7. Semi-finished product preparation and freeze-drying Take the prepared virus stock solution, determine its virus titer, add lyophilization protectant to dilute and prepare according to the preset target titer of semi-finished product (5.21gPFU / mL), and stir evenly to obtain the vaccine semi-finished product.

[0043] The semi-finished product is then packaged into vials and subjected to vacuum freeze-drying, as follows: Pre-freezing stage: The temperature of the partition is uniformly reduced to -45℃ at a rate of 1℃ / min, and kept at a constant temperature for 2.2 hours; First drying: Vacuum was applied to 16 Pa, and the temperature of the partition was increased to -15℃ at a rate of 0.5℃ / min and maintained at a constant temperature for 17 hours; Secondary drying: The vacuum level is reduced to below 3 Pa, and the temperature of the partition is increased to 27°C at a rate of 0.5°C / min and maintained at a constant temperature for 5.5 hours; After freeze-drying, the vaccine is fully stoppered under vacuum, and then capped after being removed from the box to obtain the finished varicella live attenuated vaccine.

[0044] Experimental Example 1 This experiment aims to observe the effects of the composition of the composite dissociation solution, the concentration of the enzyme, the components of the ultrasonic protectant, and the ultrasonic method on the virus harvesting effect.

[0045] 1. Experimental Grouping All groups used the same batch of MRC-5 cells and Oka strain virus to ensure consistent cell state and infection level. The groupings are shown in Table 2. Table 2 Group settings for Experiment Example 1

[0046] 2. Test Methods (1) Cell culture and virus infection: Same as steps 1-2 in Example 1, using the same batch of cells and the same batch of Oka strain virus, and all groups were operated synchronously to ensure that the cell density, infection multiple, and culture time were completely consistent.

[0047] (2) Group processing: According to the above group settings, the cells in each group were subjected to virus harvesting treatment under corresponding conditions. Three batches of independent parallel samples were prepared for each group. The cell suspensions collected from each group were filtered through a 40μm filter and the filtrate was used for subsequent detection.

[0048] (3) Detection indicators and methods: Viral titer (lgPFU / mL): The plaque method was used to determine the viral titer. The sample was serially diluted 10-fold and seeded into 6-well plates of MRC-5 cells that had grown into a dense monolayer. Two wells were seeded for each dilution, with 100 μL per well. The cells were incubated at 37°C and 5% CO2 for 7-10 days. Plaque formation was observed and the viral titer was calculated.

[0049] Residual host cell protein (ng / dose): Measured using the MRC-5 host cell protein ELISA kit, calculated per dose as 0.6 mL.

[0050] Bovine serum albumin residual amount (ng / dose): determined using a bovine serum albumin ELISA kit, with each dose calculated as 0.6 mL.

[0051] 3. Experimental Results The experimental results are shown in Table 3. The results are calculated based on the average value of three batches of parallel samples. Table 3. Effects of enzymatic hydrolysis and sonication conditions on virus harvesting efficiency.

[0052] As shown in Table 3, the group (A1) of this invention exhibits a significant advantage in controlling viral titer and host cell protein (HCP) residue. Comparing group B1 (without enzymatic dissociation) with the groups treated with enzymatic dissociation, it was found that group B1 had a high BSA residue of 4.5 ng / dose, while all groups treated with the compound enzyme had BSA levels reduced to 2.3-3.1 ng / dose. This indicates that the reduction in BSA residue is mainly attributed to the initial washing step: the compound enzyme at 20-37°C loosens and rounds the adherent cells, breaking the tight junctions between cells, allowing for more effective removal of retained bovine serum components from the culture surface during subsequent waste disposal. Simultaneously, the lowest viral titer in group B1 also reflects the greater loss of cell adhesion in the cell factory under the non-enzymatic dissociation state, resulting in a lower viral yield. Under the premise of enzymatic dissociation treatment, the viral titers of group B2 (EDTA only) and group B3 (trypsin only) were 4.88 and 4.82 lgPFU / mL, respectively, both lower than that of group A1 (5.15 lgPFU / mL). This suggests that the chelating effect of EDTA and the digestive effect of trypsin are complementary in promoting sufficient cell motility, and that the dissociation of a single group was not thorough enough, thus affecting the subsequent viral release rate.

[0053] In the concentration study, the low-concentration group (B4) had a slightly lower titer (4.95 lgPFU / mL), indicating insufficient dissociation. The high-concentration group (B5) showed a titer decrease to 4.72 lgPFU / mL, and HCP rebounded to 185 ng / dose. This suggests that excessively high enzyme concentrations may lead to over-digestion and aggregation of cells before sonication, releasing more endogenous proteins and adversely affecting viral activity.

[0054] Notably, there were significant differences in HCP residues among the groups: the B6 group without the protectant had the highest HCP at 168 ng / dose, followed by the B7 (120 ng / dose) and B8 (128 ng / dose) groups containing only a single protectant, while the A1 group had the lowest (82 ng / dose). Combined with analysis of the 40μm filtration process, the ultrasonic cavitation effect without the protectant fragmented cells into submicron-sized fragments, which penetrated the filter cartridge, leading to higher HCP levels. Simultaneously, the dual protectant increased the viral titer from 4.58 lgPFU / mL in the B6 group to 5.15 lgPFU / mL, confirming its direct protective effect on viral particle structure.

[0055] Comparing groups A1 and B9 (continuous ultrasound), all conditions were identical except for the ultrasound mode. The viral titer in group B9 decreased to 4.68 lgPFU / mL, indicating that the localized heat buildup from continuous ultrasound exceeded the protective limit of the buffer solution, leading to thermal inactivation of the virus. The intermittent ultrasound mode used in this invention provides an effective heat dissipation buffer, a necessary condition for ensuring high-titer harvesting. Comparing groups A1, A2, and A3, it was observed that within the range of 4 to 6 ultrasound cycles, neither the viral titer (5.12-5.15 lgPFU / mL) nor the residual impurities showed drastic fluctuations. This indicates that, with the synergy of the compound enzyme dissociation and the dual protective agents, this harvesting process has a strong tolerance for the number of ultrasound cycles, which is beneficial for process control and batch-to-batch consistency in actual production.

[0056] Experimental Example 2 Based on Experiment 1, this experiment aims to observe the effects of the types and proportions of each component in the freeze-drying protectant of the present invention on vaccine quality, and to complete thermal stability and long-term stability tests in accordance with the requirements of Part III of the Chinese Pharmacopoeia, so as to provide a basis for determining vaccine quality standards and shelf life.

[0057] 1. Experimental Grouping All groups were strictly controlled to maintain consistent total solids content, with only the component ratios adjusted. The groupings are shown in Table 4. Table 4 Group settings for Experiment Example 2

[0058] 2. Test Methods (1) Preparation of virus stock solution: The same batch of virus stock solution was prepared using the method in Example 1. All groups used the same virus stock solution to ensure that the initial virus titer was consistent.

[0059] (2) Preparation of protective agent: Prepare the freeze-drying protective agent for each group according to the above grouping, with 500 mL prepared for each group. The preparation method is the same as step 6 of Example 1. All groups are operated synchronously to ensure consistent environmental conditions.

[0060] (3) Preparation and freeze-drying of semi-finished products: Take the same batch of virus stock solution, determine its virus titer, and then add the protective agent of each group to dilute and prepare according to the uniform preset target titer (5.2 lgPFU / mL). Dispense the prepared semi-finished products into vials and freeze-dry them simultaneously according to the freeze-drying process in step 7 of Example 1. Prepare 3 independent parallel samples for each group.

[0061] (4) Detection indicators and methods: Basic testing: After freeze-drying, the virus titer, moisture content, endotoxin content, appearance, and reconstitution time were tested. The methods were in accordance with the requirements of the Varicella Live Attenuated Vaccine section of the Chinese Pharmacopoeia, Volume III. The experimental results are shown in Table 5.

[0062] Thermal stability test (37℃): The samples were placed in a 37℃ constant temperature chamber for 28 days. The viral titer was measured on days 0, 7, 14, 21 and 28. The focus was on whether the viral titer on day 7 met the qualified standard of ≥3.6lgPFU / mL. The experimental results are shown in Table 6.

[0063] Long-term stability test (2-8℃): The samples were placed in a cold storage at 2-8℃ for 24 months. Samples were taken in the 3rd, 9th and 18th months to test the virus titer and moisture content. Samples were taken in the 0th, 6th, 12th and 24th months for full-item testing. The focus was on whether the virus titer and moisture content met the qualified standards at 24 months. The experimental results are shown in Table 7.

[0064] Note: The qualification standards are uniformly based on the Chinese Pharmacopoeia, Volume III: virus titer ≥3.6 lgPFU / mL, moisture ≤3.0%, endotoxin ≤50 EU / dose, and all tests must meet all the requirements of the pharmacopoeia.

[0065] 3. Experimental Results Table 5. Basic test results after freeze-drying in Experiment Example 2 (average of 3 batches of parallel samples)

[0066] Table 6. Results of thermal stability test for Example 2 (lgPFU / mL, average value of 3 batches of parallel samples)

[0067] Table 7 Results of long-term stability test of Experiment Example 2 (average value of 3 batches of parallel samples placed at 2-8℃)

[0068] 4. Results Analysis Based on the basic freeze-drying results, the embodiment group of this invention exhibited extremely superior physical and chemical properties of the finished product. The viral activity remained in an extremely high range after freeze-drying, moisture control was extremely stringent, and the appearance was regular and smooth, with rapid reconstitution. In contrast, when any core component was removed from the formulation, the finished product immediately showed varying degrees of collapse, detachment, or roughness, with a significant increase in moisture content and a corresponding decrease in viral titer. Particularly when multiple key components were simultaneously missing, the freeze-dried cake even showed severe cracking, with moisture exceeding the unacceptable threshold and the titer dropping to its lowest point. This comparison strongly demonstrates that the five components in the protectant each play an irreplaceable role in constructing the freeze-drying framework and maintaining the viral spatial conformation.

[0069] When examining the influence of the proportions between components, it was found that when the proportions of sugar alcohols or amino acids deviated from the preferred range defined in this invention, even if the total solids remained unchanged, the various indicators of the lyophilized product would show a slight deterioration trend. This indicates that the components are not simply physically mixed, but rather require a specific ratio to form an optimal glassy protective network during the lyophilization process.

[0070] In the thermal stability study under simulated high-temperature stress, although the titers of all groups naturally decreased over time, the example group consistently maintained a titer level far exceeding the acceptable standard throughout the entire period, and exhibited the gentlest rate of decline. Conversely, the groups lacking any component had already been at a disadvantage from the start, and the rate of decline was further amplified under accelerated degradation, with some groups failing to meet the required test indicators. This confirms that the formulation of this invention has outstanding buffering capacity in resisting thermal stress and delaying virus inactivation.

[0071] Long-term actual storage data further solidified the above conclusions. After 24 months of storage, the viral titer in the example group remained high, moisture content showed no significant rebound, and all tests passed smoothly. In contrast, the control group, lacking the backbone components, exhibited unacceptable phenomena such as excessive moisture content or a significant drop in titer during long-term storage; while those groups with deviations in proportions barely met the standards, their final titer retention levels were significantly lower than those of the example group. In summary, the lyophilization protectant formulation and its strict component proportions established in this invention are key factors in ensuring perfect lyophilization and excellent long-term stability of the varicella live attenuated vaccine.

[0072] Experimental Example 3 This test aims to observe the differences in safety and protective effect between the protective agent of the present invention and commonly used protective agent components (dextran, human serum albumin) and published patent formulations in the prior art. All test methods and qualification standards are the same as in Test Example 2, thereby clarifying the technical advantages of the protective agent of the present invention in terms of safety and stability.

[0073] 1. Experimental Grouping The experimental groups are shown in Table 8: Table 8 Group settings for Experiment Example 3

[0074] 2. Test Methods

[0075] (1) Preparation of virus stock solution: The same batch of virus stock solution was prepared using the method in steps 1-5 of Example 1. All groups used the same virus stock solution to ensure that the initial virus titer was consistent.

[0076] (2) Preparation of protective agents: Prepare each group of freeze-drying protective agents according to the above grouping. The preparation method of D14 and D15 is the same as that of Example 1; D16 and D17 are prepared strictly according to the method disclosed in the corresponding patent. All groups are operated synchronously to ensure consistent environmental conditions.

[0077] (3) Preparation and freeze-drying of semi-finished products: Add freeze-drying protectant to each group of virus stock solution and dilute it to the preset target titer of semi-finished product (5.2 lgPFU / mL). Each group is packaged into 30 bottles, each bottle containing 0.6 mL. The freeze-drying process is carried out simultaneously according to step 7 of Example 1. Three batches of independent parallel samples are prepared for each group.

[0078] (4) Detection indicators and methods: Same as in Experiment 2, including basic indicators after freeze-drying, thermal stability at 37℃, long-term stability at 2-8℃ and moisture change, with a focus on comparing the virus activity protection effect and safety-related indicators.

[0079] 3. Experimental Results The experimental results are shown in Table 9: Table 9. Results of the comparative test of the protective agent effect in Experiment Example 3 (average value of 3 batches of parallel samples)

[0080] 4. Results Analysis As can be seen from the data comparison in Table 9 (see Experimental Example 2), the group of the present invention (C1) is at the best level among all groups in terms of initial titer, thermally accelerated stability and long-term stability after freeze-drying, and has excellent freeze-drying molding properties and reconstitution performance.

[0081] Compared to the existing macromolecular-free formulation group (D16), the titer of the present invention after freeze-drying is approximately 0.5 logarithmic orders higher. More significantly, the freeze-dried product of group D16 exhibits noticeable shrinkage and hardened clumps, with a reconstitution time as long as 28 seconds; while group C1 has a regular and smooth appearance, requiring only 15 seconds for reconstitution. This demonstrates that the multi-component synergistic system constructed from trehalose, sorbitol, amino acids, and PVP used in this invention not only completely solves the physical shaping problems of animal-free formulations, such as easy collapse and difficulty in reconstitution, but also provides excellent protection for the spatial conformation of the virus during the freeze-drying process and subsequent storage. It is noteworthy that the formulation of this invention achieves superior freeze-drying shaping and virus titer protection effects while having a significantly lower total solids content than existing formulations (D16, D17).

[0082] Compared with the control group (D14) containing dextran, the group of this invention showed significant advantages in initial titer, titer retention after 28 days at 37°C, and titer retention after 24 months at 2-8°C. Meanwhile, the D14 group exhibited slight edge detachment, while the C1 group showed excellent formation. This indicates that the present invention, by using PVP K30 instead of traditional dextran as the skeleton forming agent, avoids the potential sensitization risk of dextran while not sacrificing physical support and achieving superior long-term stable protective performance.

[0083] Compared to the control groups (D15, D17) containing human serum albumin, group C1 performed identically to the human serum albumin-containing formulation in two key indicators related to ease of clinical use: moisture control (1.5%) and reconstitution time (15 seconds). It also had a shorter reconstitution time and eliminated concerns about the safety risks associated with blood products. More importantly, in tests involving accelerated degradation at 37°C and long-term storage at 2-8°C, the viral titer in group C1 exceeded that of groups D15 and D17. These results demonstrate that this invention, through a specific synergistic ratio of L-arginine, L-histidine, and PVP, replaces the function of human serum albumin, achieving a significant improvement over existing technologies in resisting viral decay and maintaining long-term high activity.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for preparing a live attenuated varicella vaccine, characterized in that, Includes the following steps: S1: Preparation of viral stock solution, including the following steps: (1) Cell culture: MRC-5 human diploid cells were expanded and passaged within 35 generations; (2) Viral infection: After the MRC-5 human diploid cells have grown into a uniform and dense monolayer of cells, Oka strain virus is added to infect the cells for infection and culture. (3) Recombinant enzyme dissociation: After the cell pathogenesis reaches more than 70%, discard the culture medium, add the recombinant dissociation solution, and treat at 20-37℃ for 2-5 minutes to loosen the cells from the culture surface; the recombinant dissociation solution contains 0.03%-0.05% EDTA and 0.01%-0.02% recombinant trypsin by weight and volume, and the solvent is PBS buffer; (4) Protective sonication: Discard the composite dissociation solution, add buffer containing a protective agent to resuspend the cells, and perform intermittent sonication under ice bath conditions; the protective agent is trehalose at a mass-volume percentage of 2%-3% and recombinant human serum albumin at a mass-volume percentage of 0.5%-1%, and the solvent is PBS buffer; the process parameters of the intermittent sonication are: sonic power 100 W-150 W, sonication time 1-2 seconds, interval time 2-3 seconds, and total number of sonications 4-6 times; (5) Clarification and Collection: The lysate after sonication is filtered, and the filtrate is collected as the original virus solution; S2: Determine the titer of the original virus solution, add a lyophilization protectant, and dilute the virus titer to a preset target value to obtain a semi-finished product; each liter of the lyophilization protectant consists of the following components: Trehalose 45-55 g; Sorbitol 12-18 g; L-arginine 6-10 g; L-histidine 3-5 g; Polyvinylpyrrolidone 12-18 g; The remaining volume is 0.01-0.05 mol / L phosphate buffer, and the pH value of the lyophilization protectant is 7.2-7.4; S3: After repackaging the semi-finished product, freeze-dry it to obtain the live attenuated varicella vaccine.

2. The preparation method according to claim 1, characterized in that, The freeze-drying process in step S3 includes: pre-freezing to below -45°C and holding for 2-3 hours; primary drying at 10-20 Pa pressure and maintaining a partition temperature of -15°C for 15-20 hours; and secondary drying at 5 Pa pressure and maintaining a partition temperature of 25-30°C for 4-6 hours.

3. A live attenuated varicella vaccine, characterized in that, It is prepared by the method described in any one of claims 1-2.

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