Bovine virus diarrhea live vaccine based on suspension culture and preparation method thereof
By combining suspension culture technology and freeze-drying protectants, the problems of large-scale production, contamination risk, and stability in BVD vaccine production have been solved, achieving efficient, safe, and low-cost preparation of live vaccines.
Patent Information
- Application Number
- CN202511921183.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-18
- Publication Date
- 2026-03-06
AI Technical Summary
Current BVD vaccine production faces challenges such as limited large-scale production, high risk of exogenous contamination, unstable virus harvest titers, and high costs. Suspension culture technology has not yet effectively addressed issues related to BVD virus adaptation and domestication, as well as improving virus titers and freeze-drying stability under serum-free conditions.
Using suspension culture technology, attenuated BVDV strain antigen and specific freeze-drying protectants, a highly efficient and stable freeze-dried live vaccine was prepared through high-density culture in a bioreactor, serum-free or low-serum culture medium, tangential flow filtration and chromatography column purification, combined with freeze-drying and preparation of freeze-drying protectants.
This technology enables large-scale production of high-density virus culture, reduces the risk of exogenous contamination, improves virus titer stability and vaccine safety, extends shelf life, and reduces labor intensity and costs.
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Figure CN121606684A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal vaccines, and more particularly to a live bovine viral diarrhea vaccine based on suspension culture and its preparation method. Background Technology
[0002] Bovine viral diarrhea (BVD) is a serious infectious disease caused by the bovine viral diarrhea virus, which severely impacts the cattle industry. Clinical manifestations include fever, diarrhea, mucosal erosion, immunosuppression, and reproductive disorders. Currently, BVD vaccines on the market mainly include inactivated vaccines and live attenuated vaccines.
[0003] Traditional BVD vaccine production often employs adherent cell culture methods (such as MDBK cell monolayer culture), which has the following inherent drawbacks: Large-scale production is limited: the cell growth area is fixed, and large-scale production requires a large number of cell factories or roller bottles, which is cumbersome, labor-intensive, and has significant batch-to-batch differences. High risk of contamination by exogenous factors: The production process requires the use of pancreatic enzyme digestion and serum, which increases the risk of contamination by mycoplasma, exogenous viruses, etc. Virus harvest titer is unstable: it is greatly affected by cell state and inoculation time, and antigen yield and quality fluctuate greatly. High cost: It relies on imported serum and consumes a large amount of culture medium.
[0004] Suspension culture technology represents a significant innovation in animal cell culture. Cells grow freely in suspension within a bioreactor, requiring no carriers and facilitating high-density culture and process monitoring. It has been widely applied in the production of human and some veterinary vaccines. However, adapting BVD virus to suspension culture systems while maintaining its genetic stability and immunogenicity, and subsequently developing stable and highly effective live vaccines, remains a significant challenge. The main difficulties lie in: screening and acclimatizing cell lines suitable for suspension culture; optimizing the viral proliferation patterns in suspension cells; increasing viral titers under serum-free culture conditions; and maintaining the stability of the final freeze-dried live vaccine.
[0005] Therefore, this invention proposes a live bovine virus diarrhea vaccine based on suspension culture and its preparation method. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a live bovine virus diarrhea vaccine based on suspension culture and its preparation method.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: A live bovine viral diarrhea vaccine based on suspension culture, which is a freeze-dried vaccine, each dose of which contains attenuated bovine viral diarrhea virus (BVDV) antigen and freeze-dried protectant. The viral load of the attenuated BVDV strain antigen was 10. 5 -10 7 CCID50 / dose, and the BVDV attenuated strain is a cytopathic attenuated strain adapted for suspension culture; The freeze-drying protectant includes: trehalose, sucrose, gelatin hydrolysate, monosodium glutamate, and polyvinylpyrrolidone.
[0008] Preferably, the viral content of the BVDV attenuated strain antigen is 10. 6 CCID50 / head serving.
[0009] Preferably, the freeze dryer comprises components with the following final concentrations (w / v): Trehalose 3%-8%, sucrose 2%-6%, Gelatin hydrolysate 1%-3%, Monosodium glutamate 0.5%-2%, Polyvinylpyrrolidone K30 0.5%-2%.
[0010] Preferably, the attenuated BVDV strain is the Oregon C24V suspension-adapted strain or the NADL suspension-adapted strain.
[0011] A method for preparing a live bovine viral diarrhea vaccine based on suspension culture includes the following steps: S1. Suspension Cell Culture: Suspension-growth mammalian cells are seeded into a bioreactor and cultured at high density using serum-free or low-serum culture medium under controlled temperature, pH, dissolved oxygen, and stirring conditions until the cell density reaches 2.0 × 10⁻⁶. 6 - 4.0×10 6 cells / mL and viability greater than 90%; S2. Virus inoculation and proliferation: Inoculate the cultured suspension cells with a low-virulence BVDV strain at a multiplicity of infection of 0.01-0.1, and continue culturing at 33.5-34.5℃ and pH 7.0-7.2 for 48-96 hours until the viral titer reaches its peak. S3. Virus fluid harvesting and clarification: Harvest the cell-virus culture and clarify it by centrifugation or deep filtration to obtain the virus supernatant; S4. Viral fluid concentration and purification: The clarified viral fluid is concentrated 5-20 times using a tangential flow filtration system, and optionally further purified by chromatography to remove host proteins; S5. Preparation and mixing of freeze-drying protectant: Dissolve trehalose, sucrose, gelatin hydrolysate, monosodium glutamate, and polyvinylpyrrolidone K30 in water for injection, filter sterile to obtain protectant solution, mix with virus solution obtained in S3 or S4 at 2-8℃ at a volume ratio of (8-9):(2-1) to make semi-finished vaccine; S6. Packaging and freeze-drying: The semi-finished seedlings are packaged into vials, pre-frozen, dried once and dried twice, and then sealed by stoppering after drying.
[0012] Preferably, in step S1, the suspended cells are suspension-acclimated bovine kidney cells MDBK-sus or young hamster kidney cells BHK-21-sus; the culture conditions are: temperature 37.0±0.2℃, pH 7.1-7.3, dissolved oxygen DO 40%-60%, and stirring speed 50-100 rpm; in step S2, after virus inoculation, the system temperature is first lowered to 34℃ and maintained for 1-2 hours to facilitate virus adsorption, and then restored to 33.5-34.5℃ for virus proliferation culture.
[0013] Preferably, in step S3, clarification is performed by continuous flow centrifugation at 4-8°C and 6000-8000 g.
[0014] Preferably, in step S4, concentration is carried out using an ultrafiltration membrane with a molecular weight cutoff of 100-300 kDa at 4-8°C and a transmembrane pressure of 0.5-1.5 bar; purification is carried out using molecular sieve chromatography, equilibrated and eluted with phosphate buffer.
[0015] Preferably, in step S5, the lyophilized protectant solution is sterilized by a 0.22μm filter membrane; during mixing, the virus solution and the protectant solution need to be stirred and mixed at 2-8℃ to avoid generating bubbles.
[0016] Preferably, the freeze-drying step in step S6 is as follows: S61: Pre-freezing stage, cool down to -40℃ at a rate of 1℃ / min and maintain for 2-4 hours; S62: First drying stage, under a vacuum of 10-30 Pa, the temperature is raised to -20℃ and maintained for 20-30 hours; S63: Secondary drying stage, under a vacuum of 5-15 Pa, the temperature is raised to 25°C and maintained for 8-12 hours.
[0017] The beneficial effects of this invention are as follows: Advanced production process: It adopts suspension culture technology to achieve high-density cell culture and large-scale virus production in bioreactors. The process is highly controllable, with good batch-to-batch consistency, easy to scale up, and significantly reduces labor intensity and pollution risk.
[0018] Safe and pure: Serum-free or low-serum culture media can be used, reducing the risk of exogenous factor contamination from the source; combined with purification processes, host cell protein and nucleic acid residues can be effectively reduced, making the vaccine safer.
[0019] High antigen efficiency: Suspension culture facilitates efficient viral amplification in cells, resulting in a significantly higher viral titer than traditional adherent culture, thus ensuring the immunogenicity of the vaccine.
[0020] Vaccine stability: The optimized freeze-drying protectant formulation greatly improves the stability of live virus during freeze-drying and storage, extending the vaccine's shelf life. Attached Figure Description
[0021] Figure 1 This is a flowchart of a method for preparing a bovine viral diarrhea live vaccine based on suspension culture, as proposed in this invention. Detailed Implementation
[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.
[0023] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.
[0024] Example 1: Domestication and Culture of Suspension Cells Cells: Starting with adherent MDBK cells, suspension acclimation was carried out using a method of gradually reducing serum levels and gradually increasing the shaking speed. Ultimately, an MDBK-sus cell line was obtained that could stably grow in suspension in chemically defined serum-free medium (CD Serum-Free Medium) with a doubling time of approximately 24 hours.
[0025] Culture: MDBK-sus cells were seeded in 1L shake flasks containing 200mL of serum-free medium at an initial density of 3.0×10^5 cells / mL and cultured in a shaker at 37℃, 5% CO2, and 120 rpm. After 3 days, the cell density reached 2.1×10^6 cells / mL with a viability of 97%, and the cells were then used for reactor inoculation.
[0026] Reactor culture: The above cells were inoculated into a 10 L bioreactor containing 5 L of serum-free medium at an inoculation density of 5.0×10^5 cells / mL. Control conditions: temperature 37.0 °C, pH 7.2 (regulated by CO2 and 7.5% NaHCO3), DO 50%. After culturing for 48 hours, the cell density reached 2.8×10^6 cells / mL and the viability was 93%, and it was ready for virus inoculation.
[0027] Example 2: Suspension culture of virus and preparation of vaccine Virus inoculation: The BVDV Oregon C24V suspension-adapted seed virus was inoculated into the bioreactor of Example 1 at an MOI of 0.05. After virus inoculation, the temperature was adjusted to 34 °C and the stirring speed was adjusted to 30 rpm, and it lasted for 2 hours.
[0028] Virus culture: After 2 hours, the temperature was restored to 34 °C, DO 50%, and the stirring speed to 80 rpm. It was cultured for 72 hours, during which samples were taken for detection. Finally, the virus titer reached 7.5×10^7 CCID50 / mL and the cell viability decreased to 72%.
[0029] Harvest and clarification: All the cultures in the reactor were harvested and clarified by continuous flow centrifugation (4 °C, 7000 g) to obtain the virus supernatant.
[0030] Ultrafiltration concentration: Using a tangential flow ultrafiltration system with a 100 kDa MWCO, 5 L of the virus supernatant was concentrated to 500 mL.
[0031] Formulation and mixing: The lyophilization protectant was formulated at the final concentration (trehalose 5%, sucrose 4%, gelatin hydrolysate 1.5%, sodium glutamate 1%, PVP K30 1%). 500 mL of the concentrated virus solution was mixed with 55.6 mL of the 5-fold concentrated protectant solution at 4 °C until evenly mixed to make the protectant reach the working concentration, and the volume ratio of the virus solution to the protectant was approximately 9:1.
[0032] Lyophilization: Sub-packaged (2 mL / vial), and the following lyophilization procedure was carried out: ① Pre-freezing: -40 °C / 3 h; ② Primary drying: -20 °C / 25 h (20 Pa); ③ Secondary drying: 25 °C / 10 h (10 Pa). After lyophilization, the vials were stoppered and crimped.
[0033] Example 3: Inspection and evaluation of the vaccine Finished product inspection: The lyophilized vaccine prepared in Example 2 was a milky white loose mass, completely dissolved within 3 seconds after adding the diluent, and the residual moisture was 2.1%. The virus content was determined to be 3.2×10^6.5 CCID50 / dose. The sterility test, mycoplasma test, and exogenous virus test were all qualified.
[0034] Safety test: BVDV antibody-negative calves were vaccinated with 5 times the recommended dose (1 dose = 2 mL) of vaccine and observed for 14 days. No systemic reactions such as fever and diarrhea were observed, and no adverse reactions were visible at the injection site.
[0035] Efficacy test: BVDV antibody-negative calves were immunized with one dose of vaccine. Blood was collected 28 days later. The serum neutralizing antibody titer GMT reached 1:128 (log2=7). When challenged with a virulent strain, the immunized cattle were 100% protected, while all control cattle developed the disease.
[0036] Shelf life test: The vaccine was stored at 2-8℃ and -20℃, and the viral titer was monitored periodically. After 24 months of storage at 2-8℃, the viral titer decreased by less than 0.5 Log, and after 36 months of storage at -20℃, the viral titer decreased by less than 0.5 Log, indicating that the vaccine has good stability.
[0037] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A live vaccine against bovine viral diarrhea based on a suspension culture, which is a freeze-dried vaccine, characterized in that, Each dose of vaccine contains bovine viral diarrhea virus BVDV attenuated strain antigen and freeze-drying protective agent; wherein the virus content of the BVDV attenuated strain antigen is 10 5 -10 7 CCID50 / head, and the BVDV attenuated strain is a cytopathic attenuated strain adapted to suspension culture; The freeze-drying protective agent comprises trehalose, sucrose, gelatin hydrolysate, sodium glutamate, and polyvinylpyrrolidone.
2. A live vaccine against bovine viral diarrhea based on a suspension culture according to claim 1, characterized in that, The virus content of the BVDV attenuated strain antigen is 10 6 CCID50 / head.
3. The live vaccine for bovine viral diarrhea based on the suspension culture according to claim 1, characterized by that, The freeze dryer comprises the following components at the final concentration (w / v): Trehalose 3%-8%, Sucrose 2%-6%, Gelatin hydrolysate 1%-3%, Sodium glutamate 0.5%-2%, Polyvinylpyrrolidone K30 0.5%-2%.
4. The live vaccine for bovine viral diarrhea based on the suspension culture according to claim 1, characterized by, The BVDV attenuated strain is an Oregon C24V suspension adapted strain or an NADL suspension adapted strain.
5. A method for preparing a live vaccine for bovine viral diarrhea based on a suspension culture, characterized by, The method comprises the following steps: S1. Suspension cell culture: suspendably growing mammalian cells are inoculated into a bioreactor, high-density culture is carried out under the conditions of controlling temperature, pH, dissolved oxygen and stirring using serum-free or low-serum medium until the cell density reaches 2.0 x 10 6 - 4.0 x 10 6 cells / mL and the viability is greater than 90%; S2. Virus inoculation and proliferation: inoculate the BVDV attenuated strain into the cultured suspension cells at an infection multiplicity of 0.01-0.1, and continue to culture at 33.5-34.5℃ and pH 7.0-7.2 for 48-96 hours until the virus titer reaches the peak value; S3. Virus liquid harvesting and clarification: harvest the cell-virus culture, and clarify by centrifugation or depth filtration to obtain virus supernatant; S4. Virus liquid concentration and purification: concentrate the clarified virus liquid by 5-20 times using a tangential flow filtration system, and further purify by a chromatography column to remove host proteins; S5. Freeze-drying protective agent preparation and mixing: dissolve trehalose, sucrose, gelatin hydrolysate, sodium glutamate, and polyvinylpyrrolidone K30 in water for injection, obtain a protective agent solution after sterilization filtration, and mix the virus liquid obtained in S3 or S4 with the protective agent solution at a volume ratio of (8-9):(2-1) at 2-8℃ to obtain a semi-finished vaccine; S6. Sub-packing and freeze-drying: sub-pack the semi-finished vaccine into a Schlenk bottle, and perform pre-freezing, primary drying, and secondary drying, and then seal the bottle after the drying is completed.
6. The method of claim 5, wherein the live vaccine is prepared by the following steps of: In the S1 step, the suspension cells are suspension-acclimated bovine kidney cells MDBK-sus or baby hamster kidney cells BHK-21-sus; the culture conditions are as follows: temperature 37.0±0.2℃, pH 7.1-7.3, dissolved oxygen DO 40%-60%, and stirring speed 50-100 rpm; in the S2 step, after virus inoculation, the system temperature is first reduced to 34℃ and maintained for 1-2 hours to facilitate virus adsorption, and then restored to 33.5-34.5℃ for virus proliferation culture. 7. The method for preparing a bovine viral diarrhea live vaccine based on suspension culture according to claim 5, characterized in that, In the S3 step, the clarification is performed by continuous flow centrifugation under the conditions of 4-8℃ and 6000-8000 g.
8. The method for preparing a bovine viral diarrhea live vaccine based on suspension culture according to claim 5, characterized in that, In the S4 step, the concentration is performed using an ultrafiltration membrane with a molecular weight cut-off of 100-300 kDa under the conditions of 4-8℃ and transmembrane pressure 0.5-1.5 bar; the purification is performed by molecular sieve chromatography using a phosphate buffer for equilibration and elution.
9. The method for preparing a bovine viral diarrhea live vaccine based on suspension culture according to claim 5, characterized in that, In the S5 step, the freeze-drying protective agent solution is sterilized by a 0.22μm filter; when mixed, the virus liquid and the protective agent solution need to be stirred and mixed in an environment of 2-8℃ to avoid the generation of air bubbles.
10. The method for preparing a bovine viral diarrhea live vaccine based on suspension culture according to claim 5, characterized in that, The specific steps of freeze-drying in the S6 step are as follows: S61: pre-freezing stage, reduce the temperature to -40℃ at a rate of 1℃ / min and maintain for 2-4 hours; S62: primary drying stage, increase the temperature to -20℃ under a vacuum degree of 10-30 Pa and maintain for 20-30 hours; S63: Secondary drying phase, temperature ramped to 25°C and held for 8-12 hours at a vacuum of 5-15 Pa.