A freeze-dried oral attenuated live rotavirus vaccine and its preparation method and application

Abstract

The present invention provides a freeze-dried oral attenuated live rotavirus vaccine and its preparation method and application, belonging to the technical field of virus vaccine preparations. Wherein, the freeze-dried oral attenuated live rotavirus vaccine comprises a virus sterile composition; the virus sterile composition comprises RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride; the RV3 virus stock solution comprises a naturally attenuated strain RV3 virus. The freeze-dried oral attenuated live rotavirus vaccine provided by the present invention contains a naturally attenuated strain RV3 virus, and is composed of RV3 sterile powder and diluent, which is used to prevent infant diarrhea caused by rotavirus. The use of RV3 virus can extend the effective protection against rotavirus forward. The present invention improves the vaccine's ability to neutralize gastric acid through formula screening and optimization, and better protects RV3 virus from the influence of titer loss.

Description

Technical Field

[0001] The invention belongs to the technical field of virus vaccine preparations, and in particular relates to a freeze-dried oral attenuated live rotavirus vaccine and a preparation method and application thereof. Background Art

[0002] Rotavirus (RV) belongs to the Reoviridae family. It is an icosahedral RNA virus with a diameter of about 70nm and no envelope. Rotavirus has a three-layer structure (outer capsid, inner capsid and core), with a double-stranded RNA gene containing 11 segments in the middle, encoding 6 structural proteins and 6 non-structural proteins (VP1-VP4, VP6, VP7, NSP1-NSP6). Among them, the structural proteins VP4 and VP7 both have specific antigenic determinants, which can induce the production of serum-specific neutralizing antibodies and induce serum-specific protective immune responses in vivo. VP6 is the most numerous structural protein with group-specific antigenic determinants; the non-structural protein NSP4 has an enterotoxin effect.

[0003] Rotavirus is a ubiquitous pathogen. According to the serological classification method, the identified rotavirus strains can be divided into 10 groups, A, J, and A. Group A rotaviruses that cause diarrhea in infants and young animals are mainly group A, and group A rotaviruses are further divided into serotypes P (protease sensitive) and serotypes G (glycoprotein). The latest research shows that among the serotypes that have been identified to infect humans and animals, there are 32 G serotypes and 47 P serotypes, among which G1P, G2P, G3P, G4P and G9P are the main epidemic strains.

[0004] Rotavirus is the main cause of severe dehydration diarrhea in children under 5 years old worldwide. About 500,000 to 600,000 children die from rotavirus diarrhea every year, of which more than 90% of the deaths are from developing countries, causing a serious disease burden in low-income countries in Africa and Asia. Vaccination is currently recognized as one of the effective methods to prevent rotavirus infection and an important means to reduce the morbidity and mortality of rotavirus diarrhea in infants and young children.

[0005] At present, there are 6 rotavirus vaccines on the market at home and abroad, all of which are oral attenuated live vaccines; nearly 100 countries around the world have included rotavirus vaccines in their national immunization programs, but rotavirus is still one of the main causes of death in children under five years old, and most deaths occur in resource-poor environments in low- and middle-income countries with limited health care infrastructure and accessibility. According to statistics, more than 88 million infants still cannot get rotavirus vaccines. Obstacles to the global implementation of vaccines include cost, limited production capacity, poor efficacy in low-income countries, and safety issues. And the lower age limit for the rotavirus vaccines that have been launched is still 6 weeks old. Newborns are still at risk of exposure to rotavirus from birth to 6-8 weeks before the first dose of vaccine. In order to further expand the scope of immunization and reduce the risk of rotavirus infection, it is necessary to develop a vaccine that can be directly vaccinated after birth.

[0006] RV3 virus (G3P) is a naturally attenuated strain isolated from the feces of healthy newborns in Melbourne, Australia in 1977. Studies have found that newborns naturally infected with RV3 virus have cross-protection against infection with heterotypic rotavirus strains within 3 years and can prevent serious diseases. The RV3-BB vaccine prepared abroad using this virus is an oral liquid preparation. Clinical studies of this vaccine have shown that the vaccine has good safety and immunogenicity and can be used for newborns.

[0007] For oral rotavirus vaccines, the titer of rotavirus will be affected in a low pH gastric acid environment, so the ingredients of currently marketed vaccines all contain buffers to neutralize gastric acid. Considering that the gastric acid content of children under 6 months old is about 0.4mEq, and after oral vaccine, it takes about 15-30 minutes for it to pass through the stomach, during which time the amount of gastric acid secreted is about 0.4mEq.

[0008] The target neutralization capacity of the RV3-BB vaccine for gastric acid reported in the literature is 0.4mEq. Since the RV3 virus is more sensitive to acidic environments than other rotaviruses, in order to better protect the RV3 virus and avoid loss of efficacy, it is necessary to improve the vaccine's ability to neutralize gastric acid. Summary of the invention

[0009] To solve the above problems, the present invention provides a freeze-dried oral attenuated live rotavirus vaccine, comprising a virus sterile composition;

[0010] The virus sterile composition comprises RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride; the RV3 virus stock solution comprises a naturally attenuated strain RV3 virus.

[0011] Preferably, the contents of the components in the virus sterile composition in a unit dose of the freeze-dried oral attenuated live rotavirus vaccine include the following:

[0012] RV3 virus, titer ≥6.5lgFFU / dose;

[0013] Sucrose, 42mg / mL-70mg / mL;

[0014] Glycine, 25mg / mL-30mg / mL;

[0015] Polysorbate 80, 0.5mg / mL-0.7mg / mL;

[0016] Sodium chloride, 2.5mg / mL-3.6mg / mL.

[0017] Preferably, the freeze-dried oral attenuated live rotavirus vaccine further comprises a diluent;

[0018] The diluent comprises: sodium dihydrogen phosphate monohydrate, sodium citrate, sucrose and sodium hydroxide.

[0019] Preferably, the content of each component in the diluent in a unit dose of the freeze-dried oral attenuated live rotavirus vaccine includes the following:

[0020] Sodium dihydrogen phosphate monohydrate, 48mg-76mg;

[0021] Sodium citrate, 206mg-235mg;

[0022] Sucrose, 250mg / mL-350mg / mL;

[0023] Sodium hydroxide, 9.9mg / mL-11.5mg / mL.

[0024] In addition, to solve the above problems, the present invention also provides a method for preparing the freeze-dried oral attenuated live rotavirus vaccine as described in any one of the above items, comprising:

[0025] Preparing an RV3 virus stock solution, and preparing an RV3 sterile composition using the RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride;

[0026] The diluent was prepared using sodium dihydrogen phosphate monohydrate, sodium citrate, sucrose and sodium hydroxide.

[0027] Preferably, the preparation of the RV3 virus stock solution, and the preparation of the RV3 sterile composition using the RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride, comprises:

[0028] Collecting virus-carrying adherent cells and preparing the RV3 virus stock solution;

[0029] Taking the RV3 virus stock solution, adding sucrose solution, glycine solution, polysorbate 80 solution and sodium chloride solution thereto in sequence, mixing, and then sterilizing and filtering to obtain a sterile stock solution;

[0030] The sterile stock solution is freeze-dried to obtain the RV3 sterile composition.

[0031] Preferably, the collecting of virus-carrying adherent cells and preparation of the RV3 virus stock solution comprises:

[0032] Wash the cell surface with DPBS;

[0033] Based on the ratio of medium volume: sterile water for injection volume = 1: (0.2-0.8), add sterile water for injection stored at 2°C-8°C to the cell culture container;

[0034] After swelling for 15 to 30 minutes, the cell culture container is shaken. After the cells completely fall off the culture surface, the cells are harvested into a sterile container to obtain the RV3 virus stock solution.

[0035] Preferably, freeze-drying the sterile stock solution to obtain the RV3 sterile composition comprises:

[0036] The sterile stock solution is subjected to a pre-freezing treatment: the sterile stock solution is placed in a freeze dryer, the temperature is reduced from -10°C to -50°C, and then vacuumed to 15 Pa;

[0037] After the sample in the freeze dryer is dried twice under a vacuum environment, the RV3 sterile composition is obtained.

[0038] Preferably, the sample in the freeze dryer is dried twice under a vacuum environment to obtain the RV3 sterile composition, comprising:

[0039] For the first drying: the sample temperature was lowered to -45°C, the pressure was controlled at 15Pa, maintained for 120 minutes, then the temperature was raised to -26°C, the pressure was adjusted to 5Pa, maintained for 2500 minutes, then the temperature was raised to -15°C, maintained for 60 minutes;

[0040] The second drying was performed by heating the sample to 0°C, maintaining the temperature for 60 minutes, then heating it to 37°C, adjusting the pressure to 0 Pa, and maintaining the temperature for 620 minutes.

[0041] In addition, to solve the above problems, the present invention also provides a freeze-dried oral live attenuated rotavirus vaccine for use in the preparation of a medicinal product for preventing and treating rotavirus-induced diarrhea in children. The freeze-dried oral live attenuated rotavirus vaccine is the freeze-dried oral live attenuated rotavirus vaccine as described above, or the freeze-dried oral live attenuated rotavirus vaccine prepared by the preparation method described above.

[0042] The present invention provides a freeze-dried oral attenuated live rotavirus vaccine and a preparation method and application thereof, wherein the freeze-dried oral attenuated live rotavirus vaccine comprises a virus sterile composition; the virus sterile composition comprises RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride; the RV3 virus stock solution comprises a naturally attenuated strain RV3 virus. The freeze-dried oral attenuated live rotavirus vaccine provided by the present invention contains a naturally attenuated strain RV3 virus, and is composed of RV3 sterile powder and diluent, and is used to prevent infantile diarrhea caused by rotavirus. The use of RV3 virus can extend the effective protection against rotavirus forward. The present invention improves the vaccine's ability to neutralize gastric acid through formula screening and optimization, and better protects RV3 virus from the impact of titer loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The figure is a schematic diagram of the process for preparing the freeze-dried oral attenuated live rotavirus vaccine of the present invention;

[0044] Figure 2 This is a flow chart of the in vitro simulation test in Example 6 of the present invention.

[0045] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The present invention provides a freeze-dried oral attenuated live rotavirus vaccine, including a virus sterile composition;

[0048] The virus sterile composition comprises RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride; the RV3 virus stock solution comprises a naturally attenuated strain RV3 virus.

[0049] As mentioned above, according to the serological classification method, the identified rotavirus strains can be divided into 10 groups, A-J. The group A is the main cause of diarrhea in infants and young animals. The group A rotavirus is further divided into P serotype (proteasesensitive) and G serotype (glycoprotein). According to the latest research, among the serotypes that have been identified to infect humans and animals, there are 32 G serotypes and 47 P serotypes, among which G1P, G2P, G3P, G4P and G9P are the main epidemic strains.

[0050] RV3 virus is a strain used in rotavirus vaccines. Rotavirus is a type of virus that causes rotavirus infection, mainly infecting infants and young children, causing symptoms such as diarrhea and vomiting. RV3 virus strain is a strain from the human rotavirus series.

[0051] It should be noted that naturally attenuated strains refer to pathogens (such as viruses or bacteria) obtained through natural variation or sub-culture, which have relatively weak virulence and reduced pathogenicity to the host, but can still induce an effective immune response.

[0052] Naturally attenuated strains are usually produced by multiple subcultures of wild-type pathogens in vivo or in vitro. During this process, the pathogens will undergo mutations such as gene mutations or genetic recombination, resulting in a weakening of their pathogenicity. After a series of experimental screening and evaluation, the selected pathogen strains are called naturally attenuated strains.

[0053] Compared with wild-type pathogens, naturally attenuated strains have lower pathogenicity and can cause milder or no obvious disease in humans or animals, while still stimulating the immune system to produce a protective immune response. This makes naturally attenuated strains key candidates in vaccine development for the preparation of vaccines to prevent the occurrence of specific diseases.

[0054] By using naturally attenuated strains to prepare vaccines, we can reduce side effects and disease risks while ensuring immune effects, providing people with safer and more effective vaccine options.

[0055] The freeze-dried oral attenuated live rotavirus vaccine provided by the present invention contains the naturally attenuated strain RV3 virus and is composed of RV3 sterile powder and a diluent, and is used to prevent diarrhea in infants and young children caused by rotavirus. The use of RV3 virus can extend the effective protection against rotavirus. The present invention improves the vaccine's ability to neutralize gastric acid through formula screening and optimization, and better protects RV3 virus from the influence of titer loss.

[0056] Furthermore, the content of each component in the virus sterile composition, in a unit dose of the freeze-dried oral attenuated live rotavirus vaccine, includes the following:

[0057] (1) RV3 virus, titer ≥ 6.5lgFFU / dose;

[0058] (2) Sucrose, 42 mg / mL-70 mg / mL; for example, 42 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, etc.;

[0059] (3) Glycine, 25 mg / mL-30 mg / mL; for example, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, etc.;

[0060] (4) Polysorbate 80, 0.5 mg / mL-0.7 mg / mL; for example, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, etc.;

[0061] (5) Sodium chloride, 2.5 mg / mL-3.6 mg / mL. For example, it can be 2.5 mg / mL, 2.8 mg / mL, 3.0 mg / mL, 3.2 mg / mL, 3.4 mg / mL, 3.6 mg / mL, etc.

[0062] Furthermore, the freeze-dried oral attenuated live rotavirus vaccine also includes a diluent;

[0063] The diluent comprises: sodium dihydrogen phosphate monohydrate, sodium citrate, sucrose and sodium hydroxide.

[0064] Furthermore, the content of each component in the diluent in a unit dose of the freeze-dried oral attenuated live rotavirus vaccine includes the following:

[0065] Sodium dihydrogen phosphate monohydrate, 48 mg-76 mg; for example, 48 mg, 50.00 mg, 55.00 mg, 60.00 mg, 65.00 mg, 70.00 mg, 76 mg, etc.;

[0066] Sodium citrate, 206mg-235mg; for example, 206mg, 210.00mg, 215.00mg, 220.00mg, 225.00mg, 230.00mg, 235mg, etc.

[0067] Sucrose, 250 mg / mL-350 mg / mL; for example, 250 mg / mL, 300 mg / mL, 350 mg / mL, etc.;

[0068] Sodium hydroxide, 9.9 mg / mL-11.5 mg / mL. For example, it can be 9.9 mg / mL, 10.0 mg / mL, 10.2 mg / mL, 10.3 mg / mL, 10.5 mg / mL, 10.8 mg / mL, 11.0 mg / mL, 11.3 mg / mL, 11.5 mg / mL, etc.

[0069] In addition, reference Figure 1 The present invention also provides a method for preparing the freeze-dried oral attenuated live rotavirus vaccine as described in any one of the above items, comprising:

[0070] S1, preparing an RV3 virus stock solution, and preparing an RV3 sterile composition using the RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride;

[0071] S2, a diluent is prepared using sodium dihydrogen phosphate monohydrate, sodium citrate, sucrose and sodium hydroxide.

[0072] Further, the S1, prepares an RV3 virus stock solution, and uses the RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride to prepare an RV3 sterile composition, comprising:

[0073] S11, collecting virus-carrying adherent cells and preparing the RV3 virus stock solution;

[0074] S12, taking the RV3 virus stock solution, adding sucrose solution, glycine solution, polysorbate 80 solution and sodium chloride solution thereto in sequence, mixing, and then sterilizing and filtering to obtain a sterile stock solution;

[0075] S13, freeze-drying the sterile stock solution to obtain the RV3 sterile composition.

[0076] It should be noted that the addition of sucrose solution, glycine solution, polysorbate 80 solution and sodium chloride solution to the vaccine is based on the following reasons:

[0077] (1) Stability: These ingredients can enhance the stability of the vaccine and protect the active ingredients in the vaccine from light, temperature and other adverse conditions. They can provide a stable environment to prevent the degradation and ineffectiveness of the vaccine ingredients.

[0078] (2) Adjusting osmotic pressure: Sucrose solution, glycine solution and sodium chloride solution can adjust the osmotic pressure of the vaccine to make it as close as possible to the cells in the body. This helps reduce discomfort during injection and promotes the absorption and action of vaccine components.

[0079] (3) Adsorbents and emulsifiers: Polysorbate 80 solution is an adsorbent and emulsifier that helps mix and stabilize the components of the vaccine. It can help the aqueous and oil phase components to be evenly dispersed and can improve the stability and efficacy of the vaccine.

[0080] (4) Injection and vaccination routes: These solutions are also used to provide appropriate volume and capacity during injection and vaccination. They can provide the appropriate injection dose and ensure that the vaccine is effectively delivered to the human body through routes such as injection or oral administration.

[0081] In summary, ingredients such as sucrose solution, glycine solution, polysorbate 80 solution and sodium chloride solution play the roles of stability, osmotic pressure regulation, adsorption and emulsifier in the vaccine to ensure the safety, stability and effectiveness of the vaccine.

[0082] The present invention adopts a freeze-drying process when preparing the RV3 sterile composition. Compared with the traditional liquid preparation process, freeze-drying is more helpful in improving the stability of the vaccine. The production scale of the present invention is easy to expand, the raw materials of the freeze-dried protective agent and diluent used in the production are low in cost, the vaccine preparation process is simple, and the accessibility is high.

[0083] Furthermore, the step S11, collecting virus-carrying adherent cells and preparing the RV3 virus stock solution, comprises:

[0084] S111, washing the cell surface with DPBS;

[0085] S112, adding sterile water for injection stored at 2° C. to 8° C. into the cell culture container based on a ratio of culture medium volume: sterile water for injection volume = 1: (0.2-0.8);

[0086] S113, after swelling for 15 minutes to 30 minutes, shake the cell culture container, and after the cells completely fall off the culture surface, harvest the cells into a sterile container to obtain the RV3 virus stock solution.

[0087] As mentioned above, the preparation method provided by the present invention may include a method for collecting virus-carrying adherent cells, which can effectively solve the problems of introducing trypsin in conventional methods, increasing process steps, long processing time and high cost, which are not suitable for large-scale production.

[0088] Further, in S13, freeze-drying the sterile stock solution to obtain the RV3 sterile composition, comprising:

[0089] S131, pre-freezing the sterile stock solution: placing the sterile stock solution in a freeze dryer, reducing the temperature from -10°C to -50°C, and then evacuating to 15 Pa;

[0090] S132, drying the sample in the freeze dryer twice under a vacuum environment to obtain the RV3 sterile composition.

[0091] Further, in S132, the sample in the freeze dryer is dried twice under a vacuum environment to obtain the RV3 sterile composition, comprising:

[0092] S1321, perform the first drying: reduce the sample temperature to -45°C, control the pressure at 15Pa, maintain for 120 minutes, then increase the temperature to -26°C, adjust the pressure to 5Pa, maintain for 2500 minutes, then increase the temperature to -15°C, maintain for 60 minutes;

[0093] S1322, perform the second drying: heat the sample to 0°C, maintain for 60 minutes, then heat to 37°C, adjust the pressure to 0 Pa, and maintain for 620 minutes.

[0094] In addition, the present invention also provides a freeze-dried oral attenuated rotavirus live vaccine for use in the preparation of a medicinal product for preventing and treating rotavirus-induced diarrhea in children. The freeze-dried oral attenuated rotavirus live vaccine is the freeze-dried oral attenuated rotavirus live vaccine as described above, or the freeze-dried oral attenuated rotavirus live vaccine prepared by the preparation method described above.

[0095] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0096] Example 1: Collection of virus-carrying cells.

[0097] In this example, an experiment on collecting cells carrying viruses was performed.

[0098] Experimental methods:

[0099] (1) Wash the Vero cell surface with RV3 rotavirus cultured in 40 layers of cell factory once with DPBS.

[0100] (2) Add 2 L of sterile water for injection stored at 2°C-8°C according to the ratio (volume of culture medium for maintenance culture: volume of sterile water for injection for swelling = 1:0.3) and swell for 25 minutes;

[0101] (3) Gently shake the cell culture container (CF40) and wait until the cells are completely detached from the culture surface before harvesting the cells into a sterile container.

[0102] (4) Wash the cell culture container once with 2 L of 0.64% NaCl solution, collect the washing solution into the same container and mix well to complete the harvest.

[0103] A total of 10 batches of RV3 stock solution were produced using this collection method, and the results are as follows:

[0104] Table 1. Virus culture results of a single 40-layer cell factory

[0105]

[0106] Compared with the existing production methods (specific comparison items can be referred to Table 2), the method used in this embodiment for collecting virus-carrying adherent cells can effectively solve the problems of the conventional method of introducing trypsin, increasing process steps, long processing time and high cost, which are not suitable for large-scale production.

[0107] Table 2. Comparison of the effect of the method in this embodiment and the prior art in large-scale production

[0108]

[0109]

[0110] Example 2: Screening of lyoprotectant concentration.

[0111] In order to determine the potential ingredients, their concentrations and combinations that may have a significant impact on rotavirus during the freeze-drying process and storage, and to support an optimal and economical freeze-drying method, the following groups of formulations were designed for freeze-drying in this example.

[0112] 1. Experimental methods and results:

[0113] The specific information of the glycine and sucrose contents in the formula is shown in Table 3 below.

[0114] Sucrose and glycine were used as freeze-drying protective agents, and the concentrations of glycine and sucrose and the total amount of dry matter were optimized. According to the content of freeze-drying protective agents shown in the table, RV3 sterile powder semi-finished product was prepared, and after preparation, 1 mL was dispensed into vials and freeze-dried in a freeze dryer. The freeze-drying results are shown in Table 4 below.

[0115] Table 3. Freeze-drying protective agent formula

[0116] serial number Glycine content (W / V) % Sucrose content (W / V) % 1 2.5％ 3.0％ 2 2.5％ 4.2％ 3 2.5％ 5％ 4 2.5％ 7％ 5 3％ 4.2％ 6 3％ 7％ 7 4％ 3.0％ 8 4％ 4.2％ 9 4％ 6％ 10 4％ 7％

[0117] Table 4. Titer test results before and after freeze-drying

[0118]

[0119]

[0120] 2. Analysis of experimental results:

[0121] (1) The virus titer loss of samples numbered 2-6 and 8-10 were all within the qualified range (qualified product standard: virus titer loss is not higher than 0.3lgFFU / mL);

[0122] (2) The virus titer loss of samples No. 1 and 7 was greater than 0.3 lgFFU / mL, which was considered unqualified.

[0123] (3) The above results show that under the conditions of glycine concentration of 2.5%-4.0% (i.e. 25 mg / mL-40 mg / mL) and sucrose concentration of 4.2%-7.0% (i.e. 42 mg / mL-70 mg / mL), the virus titer loss before and after freeze-drying can be guaranteed to be within the acceptable range.

[0124] Example 3: Screening study of polysorbate 80 concentration.

[0125] Polysorbate 80 has the function of preventing aggregation. In this embodiment, the concentration of polysorbate 80 is investigated to ensure that the appearance of the RV3 sterile powder semi-finished product is qualified during the packaging process.

[0126] 1. Experimental methods and results:

[0127] In this embodiment, RV3 sterile powder semi-finished products containing 0.0%, 0.01%, 0.03%, 0.05% and 0.07% (W / V) polysorbate 80 were prepared, respectively. After filtration, the semi-finished products were placed at 25° C. and their appearance was observed at different time points. The specific information is shown in the following 5 tables.

[0128] Table 5. Effect of different contents of polysorbate 80 on the appearance of semi-finished products

[0129]

[0130] 2. Analysis of experimental results:

[0131] The purpose of using polysorbate 80 in this embodiment is to maintain the stability of the product appearance and ensure the stability of the product during the packaging process.

[0132] (1) Semi-finished products without polysorbate 80 will quickly produce floccules;

[0133] (2) For samples containing polysorbate 80, as the content increases, the time for the semi-finished product to maintain a clear state gradually increases;

[0134] (3) When the polysorbate 80 content is in the range of 0.05%-0.07% (i.e., 0.5 mg / mL-0.7 mg / mL), clarity can be maintained within 24 hours, which can meet the packaging requirements.

[0135] Example 4: Investigation of dilution formula.

[0136] 1. Determination of the virus's acid resistance

[0137] In this example, the lowest pH that the virus can tolerate is determined through experiments and in combination with literature.

[0138] A Experimental methods and results:

[0139] (1) Prepare buffer solutions of different pH values. Take 1 mL of virus solution and add 3 mL of buffer solution. Mix well.

[0140] (2) Take 1 mL of virus solution and mix with 3 mL of DMEM as a control.

[0141] (3) After incubation at 37°C in a water bath for 30 minutes, take a sample and measure the pH value.

[0142] (4) Take another sample and add the same volume of DMEM (containing 80 mM HEPES) to dilute it and store it at -80 °C until testing.

[0143] Table 6 below shows the virus titer detection results under different pH conditions.

[0144] Table 6. Virus titers under different pH conditions

[0145]

[0146]

[0147] B. Experimental results analysis:

[0148] (1) As shown in Table 6 above, compared with the control, when 2.92≤pH≤5.21, the virus titer of the sample was significantly reduced, and the titer loss was ≥0.5lgFFU / mL;

[0149] (2) When 5.37≤pH≤8.52, the virus titer loss of the sample is ≤0.1lgFFU / mL.

[0150] (3) The results showed that when 5.37 ≤ pH ≤ 8.52, the virus was relatively stable within 30 minutes at 37°C. Therefore, the pH of the vaccine after neutralization with gastric acid should be between 5.4 and 8.5.

[0151] 2. Determination of buffer salt concentration in dilution formula

[0152] NaH2PO4·H2O (sodium dihydrogen phosphate monohydrate) and sodium citrate are often used as components of buffer solutions, and the buffer range of NaH2PO4·H2O is 6.2-8.2, and the buffer range of sodium citrate is 4.4-6.4. The buffer ranges of the two salts are complementary, and the combination of the two salts can play a better buffering role at pH 5.4-8.5 (the stable pH range of RV3), so NaH2PO4·H2O and sodium citrate are selected as the main components of the diluent buffer salt.

[0153] A Experimental method:

[0154] Determine the amount of each component of the diluent buffer salt based on the pH value after neutralization with 8 mL of 0.1 M hydrochloric acid.

[0155] Dilutions containing different amounts of NaH2PO4·H2O and sodium citrate were prepared and neutralized with 8 mL of 0.1 M hydrochloric acid, and the pH value was measured.

[0156] Table 7. Dilution formula

[0157]

[0158] B. Experimental results analysis:

[0159] (1) As shown in the data in Table 7 above, the pH of the dilutions of Groups 1 to 3 after neutralization with 8 mL of 0.1 M hydrochloric acid was less than 5.4.

[0160] (2) When the dosage of NaH2PO4·H2O was 48-76 mg / dose and the dosage of sodium citrate was 206-235 mg / dose, the pH after neutralization with 8 mL of 0.1 M hydrochloric acid was greater than 5.4, meeting the requirements.

[0161] Example 5: Stability study after reconstitution.

[0162] 1. Experimental methods:

[0163] At different time points, take a bottle of RV3 diluent and add it to the RV3 sterile powder. After reconstitution, place it at room temperature for 0 hour, 0.5 hour, 1 hour and 2 hours, and take samples to detect the virus titer.

[0164] Table 8. Test results after different reconstitution times

[0165]

[0166] 2. Analysis of experimental results:

[0167] (1) RV3 sterile powder was reconstituted with RV3 diluent and placed at room temperature for 0, 0.5, 1, and 2 hours. The virus titer was 6.7-6.9 lg FFU / dose. Within 2 hours, the virus titer did not change significantly.

[0168] (2) After the proposed vaccine formula is reconstituted, the virus is stable at room temperature for 2 hours, meeting the requirements for use.

[0169] Example 6: In vitro simulated gastric acid test.

[0170] 1. Simulated gastric acid test group:

[0171] A Experimental method:

[0172] 1. Take 3 bottles of RV3 diluent and reconstitute 1 bottle of RV3 sterile powder. After reconstitution, mix them together as the reconstituted sample. Take the reconstituted sample for pH determination, and take another sample and temporarily store it at -80℃ for virus titer determination.

[0173] 2. Take the reconstituted sample and mix it with 8mL 0.1M hydrochloric acid (equivalent to the amount of gastric acid in the stomach of infants and young children), mix it and divide it into 3 parts. The first sample is not incubated, and the second and third samples are incubated at 37°C for 15 minutes and 30 minutes respectively. The three samples are sampled for pH determination, and the remaining samples are added with the same volume of DMEM culture medium, mixed, and temporarily stored at -80°C for virus titer detection.

[0174] 3. Control group: In the control group, the three bottles of RV3 sterile powder were reconstituted with the same volume of water, and the other 8 mL of 0.1 M hydrochloric acid was replaced with 8 mL of DMEM medium. The rest of the experimental operations, sampling points and test items were the same as those of the experimental group.

[0175] The flow chart of the in vitro simulated gastric acid test is shown in Figure 2 , the experimental results are shown in Table 9 below.

[0176] Table 9. Comparison of in vitro simulated gastric acid test results

[0177]

[0178] B. Experimental results analysis:

[0179] (1) The results are shown in the table above. The pH of the diluted solution after reconstitution was 7.4, and the pH of the water after reconstitution was 7.21. After incubation at 37°C for 0 minutes, 15 minutes, and 30 minutes after simulating gastric acid, the pH was 5.43, 5.45, and 5.42, respectively, all greater than 5.4.

[0180] (2) The virus titer after reconstitution with the diluent was 6.9 lgFFU / dose. After incubation at 37°C for 0, 15, and 30 minutes in simulated gastric acid, the virus titer was 6.8, 6.9, and 6.8 lgFFU / dose, respectively. The virus titer did not decrease significantly.

[0181] (3) The virus titer after reconstitution with water was 7.0 lgFFU / dose. After dilution with culture medium and incubation at 37°C for 0 min, 15 min, and 30 min, the virus titer was 7.0, 6.9, and 6.9 lgFFU / dose, respectively. The virus titer did not decrease significantly, and there was no significant difference in the virus titer between the control and test groups.

[0182] Experimental conclusion: RV3 sterile powder can be stably present in the stomach after being reconstituted with RV3 diluent.

[0183] Example 7: Production and preparation method of freeze-dried oral attenuated live rotavirus vaccine (Vero cells).

[0184] 1. Preparation of RV3 sterile powder

[0185] Experimental methods:

[0186] (1) Take the virus stock solution, add sucrose solution, glycine solution, polysorbate 80 solution and sodium chloride solution in sequence until the sucrose concentration is 42 mg / mL, the glycine concentration is 25 mg / mL, the polysorbate 80 concentration is 0.5 mg / mL, and the sodium chloride concentration is 3.1 mg / mL, mix, filter, and sterilize in sequence, and then dispense into 2 mL vials by a dispensing machine;

[0187] (2) Place the packaged samples in a freeze dryer and freeze-dry according to the table below;

[0188] (3) The freeze-dried sample is transferred to a capping machine for capping; after capping, the sample is inspected by light and labeled to obtain RV3 sterile powder.

[0189] Table 10. Freeze-drying parameters

[0190]

[0191]

[0192] 2. Preparation of RV3 dilution

[0193] Experimental methods:

[0194] (1) adding sodium dihydrogen phosphate monohydrate solution, sodium citrate solution, and sucrose solution in sequence, mixing well, and adjusting the pH value to 7.0-8.0 with sodium hydroxide solution;

[0195] (2) using a (0.45+0.2) μm filter to sterilize and filter to obtain a semi-finished dilution solution;

[0196] (3) The semi-finished product is divided into 2 mL vials by a filling machine and capped by a capping machine. After capping, the vials are inspected by light, labeled and packaged to obtain the RV3 diluent.

[0197] In this example, three pilot batches of RV3 sterile powder and RV3 diluent were produced, and the prepared vaccine products were tested. The results are shown in Table 11 below.

[0198] Table 11. RV3 sterile powder test results

[0199]

[0200] Table 12. RV3 dilution test results

[0201]

[0202] In summary, the freeze-dried oral attenuated live rotavirus vaccine provided by the present invention contains the naturally attenuated strain RV3 virus, and is composed of RV3 sterile powder and diluent, and is used to prevent diarrhea in infants and young children caused by rotavirus. The use of RV3 virus can extend the effective protection against rotavirus forward. The present invention improves the vaccine's neutralization ability for gastric acid through formula screening and optimization, and better protects RV3 virus from the influence of titer loss.

[0203] The method for collecting virus-carrying adherent cells used in the preparation of the present invention can effectively solve the problems of introducing trypsin in conventional methods, increasing process steps, long processing time and high cost, which are not suitable for large-scale production.

[0204] The freeze-drying process adopted by the present invention is more helpful in improving the stability of the vaccine compared with the traditional liquid preparation process; and the production scale of the present invention is easy to expand, the raw materials of the freeze-drying protectant and diluent used in the production are low in cost, the vaccine preparation process is simple, and the accessibility is high.

[0205] The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage and reaction vessel, such as the use of a continuous flow reactor, which all fall within the scope of protection of the present invention. The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage, which all fall within the scope of protection of the present invention.

Claims

1. A freeze-dried oral attenuated rotavirus live vaccine, characterized in that: including virus sterile compositions and diluents; The virus sterile composition is composed of RV3 virus stock solution, sucrose, glycine, polysorbate 80 and sodium chloride; the RV3 virus stock solution includes a naturally attenuated strain RV3 virus; the RV3 virus strain is a strain from a human rotavirus series; The diluent comprises: sodium dihydrogen phosphate monohydrate, sodium citrate, sucrose and sodium hydroxide; The content of each component in the virus sterile composition, in a unit dose of the freeze-dried oral attenuated live rotavirus vaccine, includes the following: RV3 virus, titer ≥6.5lgFFU / dose; Sucrose, 42mg / mL-70mg / mL; Glycine, 25mg / mL-30mg / mL; Polysorbate 80, 0.5mg / mL-0.7mg / mL; Sodium chloride, 2.5mg / mL-3.6mg / mL; The content of each component in the diluent, in a unit dose of the freeze-dried oral attenuated live rotavirus vaccine, includes the following: Sodium dihydrogen phosphate monohydrate, 48mg-76mg; Sodium citrate, 206mg -235mg; Sucrose, 250mg / mL-350mg / mL; Sodium hydroxide, 9.9mg / mL-11.5mg / mL.

2. The method for preparing the freeze-dried oral attenuated live rotavirus vaccine according to claim 1, characterized in that: include: Wash the cell surface with DPBS; Based on the ratio of medium volume: sterile water for injection volume = 1: (0.2-0.8), add sterile water for injection stored at 2°C-8°C to the cell culture container; After swelling for 15 to 30 minutes, the cell culture container is shaken, and after the cells completely fall off the culture surface, the cells are harvested into a sterile container to obtain the RV3 virus stock solution; Taking the RV3 virus stock solution, adding sucrose solution, glycine solution, polysorbate 80 solution and sodium chloride solution thereto in sequence, mixing, and then sterilizing and filtering to obtain a sterile stock solution; Freeze-drying the sterile stock solution to obtain the RV3 sterile composition; The diluent was prepared using sodium dihydrogen phosphate monohydrate, sodium citrate, sucrose and sodium hydroxide.

3. The method for preparing the freeze-dried oral attenuated live rotavirus vaccine according to claim 2, characterized in that: The sterile stock solution is freeze-dried to obtain the RV3 sterile composition, comprising: The sterile stock solution is subjected to a pre-freezing treatment: the sterile stock solution is placed in a freeze dryer, the temperature is reduced from -10°C to -50°C, and then vacuumed to 15 Pa; After the sample in the freeze dryer is dried twice under a vacuum environment, the RV3 sterile composition is obtained.

4. The method for preparing the freeze-dried oral attenuated live rotavirus vaccine according to claim 2, characterized in that: After the sample in the freeze dryer is dried twice under a vacuum environment, the RV3 sterile composition is obtained, comprising: For the first drying: the sample temperature was lowered to -45°C, the pressure was controlled at 15Pa, maintained for 120 minutes, then the temperature was raised to -26°C, the pressure was adjusted to 5Pa, maintained for 2500 minutes, then the temperature was raised to -15°C, maintained for 60 minutes; The second drying was performed by heating the sample to 0°C, maintaining the temperature for 60 minutes, then heating it to 37°C, adjusting the pressure to 0 Pa, and maintaining the temperature for 620 minutes.

5. Use of a freeze-dried oral attenuated live rotavirus vaccine in the preparation of a medicinal product for preventing and treating diarrhea caused by rotavirus in children, characterized in that: The freeze-dried oral live attenuated rotavirus vaccine is the freeze-dried oral live attenuated rotavirus vaccine according to claim 1, or the freeze-dried oral live attenuated rotavirus vaccine prepared by the preparation method according to any one of claims 2 to 4.

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