Vaccine lyoprotectant without animal-derived protein component and use thereof

Abstract

A vaccine lyoprotectant without an animal-derived protein component, which contains the following components: 0.2 wt.%-8wt.% of sucrose; 1.0 wt.%-10 wt.% of trehalose; 0.2 wt.%-4.0 wt.% of sodium glutamate; 0.05 wt.%-1.0 wt.% of urea; 0.05 wt.%-1.0 wt.% of arginine; and 0.05 wt.%-2.0 wt.% of glucose, with the balance being a phosphate buffer solution having a pH in the range of 6.8-7.8. The present invention further relates to a vaccine semi-finished product and a lyophilized preparation prepared from the lyoprotectant.

Description

Vaccine freeze-dried protective agent without animal-derived protein components and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a vaccine freeze-dried protective agent formula without animal-derived protein components, and application of the freeze-dried attenuated live varicella vaccine in the formula. Background Art

[0002] Varicella-zoster virus (VZV) belongs to the Herpesviridae family and the α-herpesviridae subfamily. It is the causative agent of both varicella (chickenpox) and herpes zoster (HZ). VZV is highly contagious, with a global infection rate exceeding 90%. Infection is primarily acquired through inhalation of infectious airborne droplets from patients with varicella or herpes zoster, or through contact with the virus. The typical symptom of varicella is an itchy vesicular rash accompanied by a mild fever. The disease has a distinct seasonal pattern, with prevalence in winter and spring. Morbidity is high, but mortality is low, and the prognosis is excellent. After recovery from varicella, the infected VZV remains latent in the ganglia. When immune function declines and the VZV-specific cell-mediated immunological reaction (CMIR) weakens, VZV reactivates, causing herpes zoster.

[0003] The most effective treatment for chickenpox is vaccination. Currently, the most widely used chickenpox vaccine internationally is the live attenuated varicella vaccine, which has been used for many years and has been fully proven to be safe and effective. The attenuated varicella-zoster virus strain used in the production of the live attenuated varicella vaccine is primarily the Oka strain, a WHO-recognized and widely used strain. The Oka strain was isolated and attenuated in 1974 by Dr. Takahashi of Japan. This attenuated strain was isolated from the chickenpox blister fluid of a three-year-old boy named Oka, and is the Oka strain of varicella-zoster virus. The Oka strain was cultured in human embryonic lung fibroblasts (HELF) at 34°C in Dr. Takahashi's laboratory for 11 consecutive generations; then, it was cultured in guinea pig embryo fibroblasts (GPLF) at 37°C for 12 generations to further attenuate the viral virulence; the master seed virus was prepared by two consecutive passages in WI-38 human diploid cells, and finally, the working seed virus was prepared by three consecutive passages in MRC-5 (human embryonic lung fibroblast) human diploid cells, which is the attenuated Oka strain currently widely used.

[0004] The production process of the live attenuated varicella vaccine mainly involves inoculating the attenuated varicella-zoster virus into the cell matrix, where the virus proliferates; then, the virus-infected cells are harvested, the cells are broken to release the virus, and then freeze-drying is performed after adding a freeze-drying stabilizer.

[0005] To ensure the effectiveness of the vaccine, all the live attenuated varicella vaccines on the market are freeze-dried preparations, and gelatin or human albumin and other animal-derived protein ingredients are often added to the freeze-dried preparation formula as a protective agent. Gelatin is a non-human ingredient, which is not only prone to cause allergic reactions, but also has potential safety risks of animal-derived viruses; although human albumin is a human-derived ingredient and a batch-released product, it also has safety risks of plasma-derived viruses and is expensive. The existing technology also uses dextran macromolecules to replace gelatin and human albumin preparations, but the use of dextran increases adverse reactions; or small molecule chemical components are used as protective agents, but this will affect the stability of the vaccine.

[0006] Chinese patent publication number CN105582534A discloses a lyoprotectant for a live attenuated varicella vaccine containing sucrose and sodium glutamate. However, the varicella vaccine prepared with this lyoprotectant exhibits poor stability, with viral titers significantly decreasing after 24 months at 2-8°C, approaching the lower limit of the standard.

[0007] Therefore, developing a safe, effective, stable and side-effect-reducing live attenuated varicella vaccine protectant is one of the urgent issues to be addressed in this field.

[0008] Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide a vaccine lyophilization protectant formula that does not contain animal-derived protein and does not contain macromolecular substances such as dextran, its application in the lyophilized preparation of attenuated live varicella vaccine, and the vaccine semi-finished or finished product made therefrom and the preparation method.

[0010] The first method of the present invention relates to a vaccine freeze-dried protective agent without animal-derived protein components, which comprises the following components:

[0011] Sucrose: 0.2wt.%~8wt.%;

[0012] Trehalose: 1.0 wt.% to 10 wt.%;

[0013] Sodium glutamate: 0.2wt.%~4.0wt.%;

[0014] Urea: 0.05wt.%~1.0wt.%;

[0015] Arginine: 0.05wt.%~1.0wt.%;

[0016] Glucose: 0.05wt.%~2.0wt.%;

[0017] The balance is phosphate buffer with a pH in the range of 6.8-7.8. Preferably, the vaccine is a live attenuated varicella vaccine.

[0018] In a preferred technical solution, the protective agent does not contain macromolecular chemical components with a molecular weight greater than 500, preferably does not contain macromolecular chemical components with a molecular weight greater than 400, and more preferably does not contain macromolecular chemical components with a molecular weight greater than 350; for example, dextran, etc.

[0019] In a preferred technical solution, the protective agent comprises the following components:

[0020] Sucrose: 0.4 wt.% to 7 wt.%, preferably 0.5 wt.% to 6 wt.%;

[0021] Trehalose: 1.5 wt.% to 9 wt.%, preferably 2 wt.% to 8 wt.%;

[0022] Sodium glutamate: 0.4wt.% to 3wt.%, preferably 0.5wt.% to 2wt.%;

[0023] Urea: 0.08 wt.% to 0.75 wt.%; preferably 0.1 wt.% to 0.5 wt.%;

[0024] Arginine: 0.08 wt.% to 0.75 wt.%; preferably 0.1 wt.% to 0.5 wt.%;

[0025] Glucose: 0.08 wt.% to 1.5 wt.%; preferably 0.1 wt.% to 1.0 wt.%;

[0026] The balance was phosphate buffer with a pH in the range of 7.1-7.4.

[0027] In a preferred technical solution, the protective agent consists of the following components:

[0028] Sucrose: 0.5wt.%~6wt.%;

[0029] Trehalose: 2wt.%~8wt.%;

[0030] Sodium glutamate: 0.5wt.%~2wt.%;

[0031] Urea: 0.1wt.% to 0.5wt.%;

[0032] Arginine: 0.1wt.% to 0.5wt.%;

[0033] Glucose: 0.1wt.%~1.0wt.%;

[0034] The balance was phosphate buffer with a pH in the range of 7.2-7.3.

[0035] The second aspect of the present invention relates to a method for preparing the vaccine lyoprotectant according to the first aspect of the present invention, comprising the following steps: weighing each component according to its content, fully dissolving the components in an appropriate amount of phosphate buffer, fixing the volume and adjusting the pH, and sterilizing by autoclaving or filtration. Alternatively, weighing each component in sequence according to its content, fully dissolving each component in an appropriate amount of phosphate buffer, mixing, fixing the volume and adjusting the pH, and sterilizing by autoclaving or filtration. Preferably, the vaccine is a live attenuated varicella vaccine.

[0036] The third aspect of the present invention relates to the use of the vaccine lyoprotectant according to the first aspect of the present invention in the preparation of a vaccine. Preferably, the vaccine is a live attenuated varicella vaccine.

[0037] The fourth aspect of the present invention relates to a semi-finished vaccine product, comprising a certified vaccine stock solution and the vaccine freeze-dried protective agent according to the first aspect of the present invention. Preferably, the vaccine is a live attenuated varicella vaccine.

[0038] A fifth aspect of the present invention relates to a lyophilized vaccine formulation comprising a certified vaccine stock solution and the vaccine lyoprotectant according to the first aspect of the present invention. Preferably, the vaccine is a live attenuated varicella vaccine. The lyophilized vaccine formulation is prepared by mixing and lyophilizing the certified vaccine stock solution and the vaccine lyoprotectant according to the first aspect of the present invention.

[0039] A sixth aspect of the present invention relates to a method for preparing a freeze-dried vaccine formulation according to the fifth aspect of the present invention, comprising the steps of diluting a qualified vaccine stock solution with the vaccine freeze-dried protectant according to the first aspect of the present invention based on viral titer, and then packaging and freeze-drying the solution. Preferably, the vaccine is a live attenuated varicella vaccine, and more preferably, the vaccine virus titer is ≥2000 PFU / ml, for example, 3000 PFU / ml or 4000 PFU / ml.

[0040] Beneficial effects of the present invention:

[0041] 1. The vaccine freeze-dried protective agent of the present invention does not contain animal-derived (including human-derived) proteins, such as gelatin, human serum albumin, etc., and does not contain macromolecular substances such as dextran, and the components contained are all small molecular substances; since the freeze-dried protective agent of the present invention does not contain macromolecular allergenic components, the vaccine is safer and less likely to cause allergic reactions. It is a new generation of freeze-dried protective agent and can be used for freeze-drying of live attenuated varicella vaccine.

[0042] 2. The live attenuated varicella vaccine prepared using the vaccine freeze-dried protective agent of the present invention has good stability and low cost. DETAILED DESCRIPTION

[0043] The present invention is further explained below with reference to specific examples so that those skilled in the art can better understand the advantages and technical effects of the present invention. However, it should be understood that these examples are only for the purpose of more detailed and specific description and should not be construed as limiting the present invention in any form.

[0044] In the present invention, unless otherwise specified, all operations are carried out at room temperature and normal pressure; the materials and operating methods used in the present invention are well known in the art; the instruments and equipment used in the following examples are all conventional equipment in the art; the culture media used are all commercially available conventional culture media, and those skilled in the art are familiar with the components and contents thereof.

[0045] Example 1: Preparation of live attenuated varicella vaccine stock solution and vaccine freeze-dried preparation

[0046] The production process of the live attenuated varicella vaccine involves inoculating the attenuated varicella-zoster virus into the cell matrix, where the virus proliferates. The virus-infected cells are then harvested, disrupted to release the virus, and then freeze-dried after adding a lyoprotectant. The specific process is as follows:

[0047] Preparation of varicella live attenuated vaccine stock solution:

[0048] (1) The virus strain used was the attenuated Oka strain of varicella-zoster virus, and the culture medium was MRC-5 human embryonic lung diploid cells;

[0049] (2) MRC-5 human embryonic lung diploid cells were serially passaged at a culture temperature of 37±1°C and cultured to no more than 41 generations using MRC-5 cell culture medium; the MRC-5 cell culture medium was MEM growth medium with a pH value of 7.0 to 7.8 and containing 5 to 25% calf serum;

[0050] (3) Viral infection: After the MRC-5 human embryonic lung diploid cells have grown to a monolayer, the supernatant is removed and the Oka strain of varicella virus is directly added at an MOI of 0.001 to 0.1 to infect the MRC-5 human embryonic lung diploid cells. The cells are adsorbed at 36 ± 1°C for ≥ 1 h. Then, maintenance medium (MEM + 2 to 10% bovine serum) is added and the cells are cultured at 36°C.

[0051] (4) 3 to 5 days after infection, when the cell lesion rate reaches ≥50%, the cells are harvested and the MRC-5 cell culture medium is discarded. The surface of the MRC-5 human embryonic lung diploid cells is washed with Earle's solution equivalent to more than twice the MRC-5 cell culture medium to remove bovine serum. The MRC-5 human embryonic lung diploid cells are digested with 0.03 to 0.05% EDTA solution and centrifuged at 2000 rpm for 8 to 10 minutes at 2 to 8°C. The supernatant is discarded and the precipitate is retained. The vaccine freeze-dried protective agent is added to the precipitate and resuspended. The cells containing varicella virus are collected, sampled for sterility inspection, and stored at -70°C.

[0052] (6) The cell suspension containing varicella virus that has passed the sterility test in step (5) is combined, ultrasonically disrupted at 20-30 kHz for 1-3 minutes at 2-8°C, and the sample is sampled for stock solution testing and then stored at -70°C;

[0053] (7) The vaccine was tested in accordance with the monograph on live attenuated varicella vaccine in the 2020 edition (Volume III) of the Pharmacopoeia of the People's Republic of China, and all met the requirements.

[0054] Preparation of varicella live attenuated vaccine freeze-dried preparation: After the varicella live attenuated vaccine stock solution is qualified, the vaccine freeze-drying protective agent of the present invention is added for dilution and mixed evenly. The virus titer is 80,000 PFU / ml, which becomes a vaccine semi-finished product, and is immediately packaged according to the specification of 0.5 ml / vial. The packaged varicella live attenuated vaccine semi-finished product is then subjected to a freeze-drying process to obtain a varicella live attenuated vaccine freeze-dried preparation; the freeze-drying adopts a low-temperature vacuum freeze-drying method. After the semi-finished product is packaged and packed, the freeze drying box is pre-frozen to -45°C, so that the product temperature reaches below -45°C and is maintained for at least 2 hours. The vacuum is turned on to enter the first stage of drying, i.e., sublimation drying. The vacuum control is set to 20Pa±5Pa. The temperature of the heat transfer oil is first set to -20°C and maintained for 8 hours. Then, the second stage of drying, i.e., desorption drying, is entered. The initial temperature of the heat transfer oil is set to 35°C. After maintaining for 6 hours, the freeze-drying is completed to prepare the varicella live attenuated vaccine freeze-dried preparation.

[0055] Example 2: Preparation of three different formulations of animal-free protein lyophilization protectants

[0056] Three lyoprotectant formulations were prepared by weighing the components according to their respective weight percentages listed in Table 1 and mixing them. The remainder was 0.01 mol / L phosphate buffer at pH 7.2. Simultaneously, a control lyoprotectant formulation was prepared based on the formulation disclosed in Chinese patent application publication number CN105582534A (0.01% sodium glutamate and 5% sucrose) (see Comparative Example 2 below for details).

[0057] Table 1: Lyoprotectant formulations

[0058] Example 3: Protective Effect of Animal-Free Protein Lyoprotectant on Live Attenuated Varicella Vaccine (Accelerated Stability)

[0059] The three protective agents of Example 2 and the control protective agent were prepared, and the lyophilized formulation of the live attenuated varicella vaccine was prepared according to the process of Example 1. The obtained vaccine was placed at 37° C. for accelerated stability study of the product.

[0060] Table 2: Accelerated stability of live attenuated varicella vaccine (37°C)

[0061] These accelerated stability results demonstrate that the freeze-dried live attenuated varicella vaccine prepared with the three lyoprotectant formulations exhibited excellent accelerated stability, with all test parameters meeting the requirements at different time points. The viral titer decline trends for the freeze-dried live attenuated varicella vaccine prepared with the three lyoprotectant formulations were essentially consistent. After 7 days of accelerated testing, the titer showed no significant decrease; after 28 days of accelerated testing, the titer, while still somewhat decreased, still met the standard requirements. The control formulation exhibited a significant titer decrease, and after 28 days of accelerated testing, the titer was already below the standard requirement (3.3 lgPFU / dose).

[0062] Example 4: Protective Effect of Animal-Free Protein Lyophilization Protectant on Live Attenuated Varicella Vaccine (Long-Term Stability)

[0063] The three protective agents of Example 2 and the control protective agent were prepared, and the lyophilized preparation of the live attenuated varicella vaccine was prepared according to the process of Example 1. The obtained vaccine was placed at 2-8°C for long-term stability study.

[0064] Table 3: Long-term stability of live attenuated varicella vaccine (2-8°C)

[0065] NA: Not applicable

[0066] These long-term stability results demonstrate that the freeze-dried live attenuated varicella vaccine formulated with the three lyoprotectant formulations exhibited excellent long-term stability over a 24-month period, with all test parameters meeting quality requirements at different time points. The virus titers of the freeze-dried live attenuated varicella vaccine formulated with the three lyoprotectant formulations did not decrease significantly at 24 months, meeting quality requirements. The titer of the control formulation decreased significantly, reaching the standard requirement (3.3 lgPFU / dose) by 24 months.

[0067] Comparative Example 1

[0068] Chinese patent application publication number CN105582534A discloses a lyoprotectant for a live attenuated varicella vaccine, which contains sucrose and sodium glutamate, and is used for the preparation of a live attenuated varicella vaccine. This example uses the vaccine lyoprotectant in CN105582534A, which does not contain gelatin, human serum albumin, and dextran, as a comparative lyoprotectant 2 (as described in Examples 2-4), to prepare a freeze-dried live attenuated varicella vaccine (according to the relatively preferred formulation B of the patent application), and the specific steps are as follows:

[0069] Comparative lyophilization protective agent 2: 0.01% sodium glutamate and 5% sucrose, the balance being 0.01 mol / L phosphate buffer at pH 7.2. The preparation was carried out using the same method as in Example 2 of the present application.

[0070] The same batch of varicella live attenuated vaccine stock solution prepared according to the method for preparing varicella live attenuated vaccine stock solution in Example 1 of the present application was taken. After passing the test, the comparison freeze-drying protectant 2 and the vaccine freeze-drying protectants of the three formulas of the present invention were added for dilution and mixed evenly. The virus titer was 80,000 PFU / ml to become a vaccine semi-finished product, and immediately packaged according to the specification of 0.5 ml / piece. The packaged varicella live attenuated vaccine semi-finished product was then subjected to a freeze-drying process to obtain a varicella live attenuated vaccine freeze-dried preparation; the freeze-drying method adopted was a low-temperature vacuum freeze-drying method. After the semi-finished product was packaged and packed, the freeze drying oven was pre-frozen to -45, so that the product temperature reached below -45°C and was maintained for at least 2 hours. The vacuum was turned on to enter the first stage of drying, i.e., sublimation drying. The vacuum control was set to 20 Pa ± 5 Pa, and the heat transfer oil temperature was first set to -20°C and maintained for 8 hours. Then, the second stage of drying, i.e., desorption drying, was entered. The initial temperature of the heat transfer oil was set to 35°C. After maintaining for 6 hours, the freeze-drying was completed to prepare the varicella live attenuated vaccine freeze-dried preparation.

[0071] Comparison of the obtained lyophilized preparations of the live attenuated varicella vaccine shows that the live attenuated varicella vaccine prepared using the comparative lyophilized protective agent 2 has poor stability. After 28 days at 37°C, the virus titer decreased significantly and was already lower than the standard requirement (3.31 g PFU / dose); after 24 months at 2-8°C, the virus titer decreased significantly, basically approaching the lower limit of the standard requirement (3.31 g PFU / dose). The live attenuated varicella vaccine prepared using the vaccine lyophilized protective agent of the present invention that does not contain dextran, gelatin, or human serum albumin has a clear composition and good stability. After 24 months, the virus titer did not decrease significantly, and other indicators also met the requirements.

[0072] In summary, the animal-derived protein-free freeze-dried protective agent formula of the present invention is suitable for the live attenuated varicella vaccine. The freeze-dried preparation of the live attenuated varicella vaccine prepared therefrom has good stability, does not contain large molecular allergenic components, reduces side effects, and has good safety.

[0073] The scope of protection of the present invention shall be subject to the definition of the claims. The embodiments are merely illustrative of the concepts and principles of the present invention. It should be understood that these specifically described embodiments should not constitute a limitation on the scope of the present invention.

Claims

1. A vaccine freeze-dried protective agent without animal-derived protein components, characterized in that: It contains the following components: Sucrose: 0.2wt.%~8wt.%; Trehalose: 1.0wt.%~10wt.%; Sodium glutamate: 0.2wt.%~4.0wt.%; Urea: 0.05wt.%~1.0wt.%; Arginine: 0.05wt.%~1.0wt.%; Glucose: 0.05wt.%~2.0wt.%; The balance was phosphate buffer with a pH in the range of 6.8-7.

8.

2. The vaccine freeze-dried protective agent according to claim 1, characterized in that The protective agent does not contain any macromolecular chemical component with a molecular weight greater than 500, preferably does not contain any macromolecular chemical component with a molecular weight greater than 400, and more preferably does not contain any macromolecular chemical component with a molecular weight greater than 350.

3. The vaccine freeze-dried protective agent according to claim 1 or 2, characterized in that: It contains the following components: Sucrose: 0.4 wt.% to 7 wt.%, preferably 0.5 wt.% to 6 wt.%; Trehalose: 1.5 wt.% to 9 wt.%, preferably 2 wt.% to 8 wt.%; Sodium glutamate: 0.4wt.% to 3wt.%, preferably 0.5wt.% to 2wt.%; Urea: 0.08 wt.% to 0.75 wt.%; preferably 0.1 wt.% to 0.5 wt.%; Arginine: 0.08 wt.% to 0.75 wt.%; preferably 0.1 wt.% to 0.5 wt.%; Glucose: 0.08 wt.% to 1.5 wt.%; preferably 0.1 wt.% to 1.0 wt.%; The balance was phosphate buffer with a pH in the range of 7.1-7.

4.

4. The vaccine freeze-dried protective agent according to claim 3, characterized in that It consists of the following components: Sucrose: 0.5wt.%~6wt.%; Trehalose: 2wt.%~8wt.%; Sodium glutamate: 0.5wt.%~2wt.%; Urea: 0.1wt.%~0.5wt.%; Arginine: 0.1wt.%~0.5wt.%; Glucose: 0.1wt.%~1.0wt.%; The balance was phosphate buffer with a pH in the range of 7.2-7.

3.

5. The method for preparing a vaccine freeze-dried protective agent according to any one of claims 1 to 4, comprising the following steps: The components are weighed according to their contents, fully dissolved in a suitable amount of phosphate buffer, fixed to volume and adjusted to pH, and sterilized by high pressure or filtration to obtain the product; or the components are weighed in sequence according to their contents, and each component is separately fully dissolved in a suitable amount of phosphate buffer, mixed, fixed to volume and adjusted to pH, and sterilized by high pressure or filtration to obtain the product.

6. Use of the vaccine lyophilization protectant according to any one of claims 1 to 4 in the preparation of vaccines.

7. A vaccine semi-finished product comprising a vaccine stock solution and a vaccine lyophilization protectant according to any one of claims 1 to 4.

8. A vaccine freeze-dried preparation, comprising a vaccine stock solution and the vaccine freeze-dried protective agent according to any one of claims 1 to 4.

9. A method for preparing the freeze-dried vaccine preparation according to claim 8, characterized in that: It includes the following steps: The qualified vaccine stock solution is diluted with the vaccine freeze-drying protective agent according to any one of claims 1 to 4 according to the virus titer, and then packaged and freeze-dried to obtain the vaccine.

10. The vaccine lyoprotectant according to any one of claims 1 to 4, or the method according to claim 5, or the use according to claim 6, or the vaccine semi-finished product according to claim 7, or the vaccine lyophilized preparation according to claim 8, or the method according to claim 9, characterized in that: The vaccine is a live attenuated varicella vaccine.

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