The process of preparing, filling, and freeze-drying a live vaccine against porcine reproductive and respiratory syndrome virus

Abstract

The present invention discloses a process for formulating, filling and freeze-drying a live PRRS virus vaccine. 1) Take out the PRRS virus antigen stored at -40°C, melt it by gradient cooling, then transfer it to the antigen transfer room. After shaking the antigen evenly, transfer it to the formulation tank through a pipeline, and start the agitator paddle in the formulation tank to further mix it evenly; 2) Inject the protective agent into the formulation tank through a pipeline, stir while injecting. When the required dose of the protective agent is added, close the valve, continue to stir for 3-4 minutes, adjust the pH of the suspension to 6.5-6.8, continue to stir evenly, and transfer the prepared finished product to the filling system through a pipeline; 3) Use a fully automatic filling system for filling; 4) Subject the filled product to pre-freezing, sublimation drying and desorption drying to obtain the live PRRS virus vaccine product. The live PRRS virus vaccine product produced by the process of the present invention can better control the residual moisture of the product. The product is in the form of a light yellow spongy and porous mass, is easily soluble, and has very little potency loss within the validity period of 18 months.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biological products, and particularly to a preparation, filling and freeze-drying process for a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV). Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is a highly contagious disease mainly characterized by reproductive disorders in sows, and respiratory symptoms in piglets and fattening pigs. Diseased pigs, virus-contaminated aerosols, vector insects, birds, and transportation tools can all cause the transmission of PRRSV. At present, the prevention and control of porcine reproductive and respiratory syndrome still mainly rely on vaccine immunoprophylaxis. The freeze-dried live vaccine against PRRSV is a relatively effective vaccine, which can effectively stimulate the pig body to produce strong and lasting immunity, control the severity of the disease, reduce the clinical incidence rate, and also improve production performance such as the daily weight gain of pigs.

[0003] The freeze-dried live vaccine against PRRSV is prepared by expanding the culture of PRRSV to a certain antigen amount, adding a freeze-drying protectant for formulation, filling according to requirements, and increasing the vacuum degree in the freeze-dryer through low-temperature freezing. The water in the antigen will be separated by sublimation, and after sublimation and desorption drying treatment, a dry powder maintaining the original virus immunogenicity is prepared. When in use, it only needs to be completely diluted with the corresponding diluent, and then immunization can be carried out. This vaccine has a small dosage, is convenient to store, and can quickly generate immune protection.

[0004] At present, the freeze-dried live vaccine against PRRSV is widely used in the market, but there are still the following problems: (1) The solubility of the vaccine is unstable. Some batches can be dissolved by standing for 3 seconds after adding the diluent, while some require a longer time to dissolve, affecting the use efficiency of the vaccine. (2) The appearance of the freeze-dried vaccine is defective and incomplete, and some show shrinkage and sidewall collapse. (3) After freeze-drying, the antigen titer loss is relatively large. (4) The remaining moisture in the finished product is unstable and easily exceeds the standard. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a preparation, filling and freeze-drying process for a live vaccine against PRRSV, aiming to improve the vaccine solubility, enhance the physical properties, reduce the moisture content of the finished product, and reduce the loss of antigen titer.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A preparation, filling and freeze-drying process for a live vaccine against PRRSV includes the following steps:

[0008] 1) Antigen gradient cooling and melting

[0009] Take out the porcine reproductive and respiratory syndrome virus antigen stored at -40°C, place it in a -20°C cold storage for 24 - 36 hours, then transfer it to a 2 - 8°C cold storage for 15 - 24 hours. Then thaw the antigen on a large-capacity shaker with the shaker temperature set at 22 - 25°C. When the antigen is completely melted and there is no longer a large amount of condensed water on the bottle wall, and at this time the antigen is about 6 - 10°C, the antigen can be taken out of the shaker, transferred to the antigen transfer room, shaken well and then transmitted to the vaccine preparation tank through a pipeline; in the vaccine preparation tank, start the agitator paddle to further mix evenly;

[0010] 2) Vaccine preparation

[0011] Adjust the rotation speed of the agitator paddle in the vaccine preparation tank to 120 - 150 r / min. Pump the protectant kept at 37°C in the protectant preparation tank into the vaccine preparation tank through a pipeline, stirring while pumping. When the required dose of the protectant is added, close the valve and continue stirring for 3 - 4 minutes. Adjust the pH of the suspension to 6.5 - 6.8 (preferably 6.6), and continue stirring for 10 - 12 minutes until it is evenly stirred. Then pump the prepared finished product into the filling system through a pipeline;

[0012] 3) Filling:

[0013] Use a fully automatic filling system for filling;

[0014] This system consists of two parts: filling and weighing. Each filling pump is controlled by an independent servo motor. The filling program can automatically adjust the stroke of the servo motor, thereby adjusting the filling volume; before filling starts, set the filling weight in the weighing system and start automatic weighing. When all filling pumps are adjusted to the required filling volume, filling officially begins. During filling, the automatic weighing system will regularly sample and check whether the filling volume is accurate according to the set frequency to ensure the stability of the filling volume of this batch;

[0015] 4) Freeze-drying

[0016] 4-1) Pre-freezing:

[0017] Put the filled product into the chamber at 18 - 26°C. When the temperature of the product drops to -4 - 0°C, then raise the temperature to 18 - 20°C and keep it warm for 50 - 60 minutes. Then continue to cool down to -40 - -38°C and keep it warm for 250 - 300 minutes;

[0018] 4-2) Sublimation drying:

[0019] After pre-freezing, evacuate to vacuum. When the vacuum drops to 28 - 30 Pa, start heating. The shelf temperature is -5 - 3°C and the vacuum degree does not exceed 25 Pa. Maintain this vacuum degree and keep it warm for 400 - 700 minutes;

[0020] 4-3) Desorption drying:

[0021] The product obtained by sublimation drying is heated to 20 - 28°C, the vacuum degree in the chamber is reduced to 0.3 - 5 Pa, and this vacuum degree is maintained and heat-insulated for 200 - 300 min.

[0022] The present invention has the following beneficial effects:

[0023] 1. After the antigen is harvested, it is stored in a cold storage at -40°C. Before preparing the vaccine, it is melted by a gradient cooling method, which can reduce the loss of antigen titer during storage and the rupture of the antigen storage container during the vaccine melting process.

[0024] 2. In the vaccine melting process, after the antigen melts until the ice cubes completely disappear, it still needs to be warmed back to 6 - 10°C on a shaker. The protective agent needs to be kept at 37°C. This method can reduce the temperature difference between the antigen and the protective agent, which is beneficial to the uniform mixing of the protective agent and the antigen, and reduce the loss caused by uneven products affecting freeze-drying. If the antigen is just melted and formulated with the protective agent, the antigen temperature is relatively low. After the colloidal substance of the protective agent is mixed with the antigen, its fluidity is poor when it meets cold, and it is difficult to mix evenly.

[0025] 3. After formulating the vaccine, the pH of the mixture of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and the protective agent (hereinafter referred to as the suspension) is adjusted to 6.5 - 6.8 to ensure the stability of the PRRSV titer.

[0026] 4. During filling, a stirring paddle is installed at the bottom of the vaccine formulation tank and stirred continuously. The filling process uses an automatic weighing device to monitor the filling accuracy regularly, which increases the uniformity of the product during the filling process and is convenient for the applicability of freeze-drying parameters to the freeze-drying of vaccines in different batches.

[0027] 5. In the freeze-drying process, the products enter the chamber at room temperature. After all the products are filled, the temperature of the shelves starts to be reduced. During the temperature reduction process, annealing treatment is carried out. This step can reduce the phenomenon of side wall collapse of the products. Annealing means that the products are first cooled and then heated, and then cooled again to a certain temperature to ensure that all products reach the target temperature.

[0028] 6. The pre-freezing time should be sufficient to ensure that the products are frozen solid; the sublimation drying and desorption drying should be maintained for a long enough time, and the remaining moisture of the products is controlled at about 2.4 - 2.9%.

[0029] 7. The live PRRSV vaccine products produced by the freeze-drying process of the present invention can better control the remaining moisture of the products. The appearance of the products is a light yellow spongy porous mass, which is easily soluble, and the titer loss is extremely small within the 18-month validity period. Specific embodiments

[0030] Example 1

[0031] The titer loss after lyophilization is relatively large compared to the titer of the previous antigen. Most of the loss is not directly caused by the lyophilization process, but rather by the storage conditions of the antigen, resulting in titer depletion. Therefore, the storage conditions of the antigen are particularly important.

[0032] Table 1 Changes in the titer of porcine reproductive and respiratory syndrome virus (PRRSV) antigen under storage conditions of -20°C and -40°C

[0033]

[0034]

[0035] Table 1 shows that compared with storing the antigen at -20°C, the titer of the antigen stored at -40°C is more stable and the loss is smaller.

[0036] Example 2

[0037] The virulence of the porcine reproductive and respiratory syndrome virus (PRRSV) antigen is stable when the pH value is between 6.5 and 7.5. To determine the optimal pH of the suspension during vaccine formulation, the following experiment was conducted in this invention;

[0038] Three batches of porcine reproductive and respiratory syndrome virus (PRRSV) antigen were mixed with the protective agent to obtain suspensions, namely Test Group I, Test Group II, and Test Group III. Each group of suspensions was divided into 6 small vials, adjusted to 6 different pH values, and then lyophilized in the same lyophilization cabinet. After lyophilization, the titer was measured uniformly. The test results are shown in Table 2.

[0039] Table 2 Changes in the titer of suspensions with different pH values after lyophilization

[0040]

[0041] Table 2 shows that as the pH value of the suspension increases, the titer loss of the lyophilized suspension also gradually increases. When the pH value of the suspension is 6.6, the titer loss after lyophilization is the least. Therefore, the optimal pH of the suspension during vaccine formulation is 6.6.

[0042] Example 3

[0043] A process for formulating, filling, and lyophilizing a live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine, comprising the following steps:

[0044] 1) Gradient cooling and thawing of the antigen

[0045] Take out the porcine reproductive and respiratory syndrome virus (PRRSV) antigen stored at -40°C, place it in a -20°C cold storage for 30 h, then place it in a 5°C cold storage for 20 h. Then thaw the antigen on a large-capacity shaker, with the shaker temperature set at 23.5°C. When the antigen is completely thawed and there is no longer condensed water on the bottle wall, and the antigen is about 8°C at this time, the antigen can be taken out of the shaker, transferred to the antigen transfer room, shaken well, and then transferred to the vaccine formulation tank through a pipeline; start the agitator paddle in the vaccine formulation tank to further mix evenly;

[0046] 2) Vaccine preparation

[0047] Adjust the rotation speed of the stirring paddle in the vaccine preparation tank to 135 r / min. Pump the protective agent kept at 37°C in the protective agent preparation tank into the vaccine preparation tank through a pipeline while stirring. When the required dose of the protective agent is added, close the valve and continue stirring for 3.5 min. Adjust the pH value of the suspension to 6.6 and continue stirring for 11 min. Then pump the prepared finished product into the filling system through a pipeline;

[0048] 3) Filling:

[0049] Use a fully automatic filling system for filling;

[0050] 4) Freeze-drying

[0051] 4-1) Pre-freezing:

[0052] Put the filled product into the drying cabinet at 22°C. When the temperature of the product drops to -2°C, raise the temperature to 19°C and keep it warm for 55 min. Then continue to cool it to -39°C and keep it warm for 280 min;

[0053] 4-2) Sublimation drying:

[0054] After pre-freezing, evacuate the air. When the vacuum drops to 29 Pa, start heating. The temperature of the shelf is 0°C and the vacuum degree does not exceed 25 Pa. Maintain this vacuum degree and keep it warm for 550 min;

[0055] 4-3) Desorption drying:

[0056] Raise the temperature of the product obtained by sublimation drying to 24°C. The vacuum degree in the cabinet drops to 2.5 Pa. Maintain this vacuum degree and keep it warm for 250 min.

[0057] Example 4

[0058] A process for preparing, filling and freeze-drying a live vaccine against porcine reproductive and respiratory syndrome virus, comprising the following steps:

[0059] 1) Antigen gradient cooling and thawing

[0060] Take out the porcine reproductive and respiratory syndrome virus antigen stored at -40°C, put it in a cold storage at -20°C for 24 h, then put it in a cold storage at 2°C for 15 h. Then place the antigen on a large-capacity shaker for thawing. The temperature of the shaker is set at 22°C. When the antigen is completely thawed and there is no more condensed water on the bottle wall, and the temperature of the antigen is about 6°C at this time, take out the antigen from the shaker, transfer it to the antigen transfer room, shake the antigen well and transfer it to the vaccine preparation tank through a pipeline; Start the stirring paddle in the vaccine preparation tank to further mix it evenly;

[0061] 2) Vaccine preparation

[0062] Adjust the rotation speed of the stirring paddle in the seedling preparation tank to 120 r / min. Pump the protectant kept at 37°C in the protectant preparation tank into the seedling preparation tank through a pipeline while stirring. After the protectant is added to the required dosage, close the valve and continue stirring for 3 minutes. Adjust the pH of the suspension to 6.6, continue stirring for 10 minutes, and pump the prepared finished product into the filling system through a pipeline;

[0063] 3) Filling:

[0064] Use a fully automatic filling system for filling;

[0065] 4) Freeze-drying

[0066] 4-1) Pre-freezing:

[0067] Put the filled product into the drying cabinet at 18°C. When the temperature of the product drops to -4°C, raise the temperature to 18°C and keep it warm for 50 minutes, then continue to cool down to -40°C and keep it warm for 250 minutes;

[0068] 4-2) Sublimation drying:

[0069] After pre-freezing, evacuate the air. When the vacuum drops to 28 Pa, start heating. The temperature of the shelf is -5°C, and the vacuum degree does not exceed 25 Pa. Maintain this vacuum degree and keep it warm for 400 minutes;

[0070] 4-3) Desorption drying:

[0071] Raise the temperature of the product obtained by sublimation drying to 20°C. The vacuum degree in the drying cabinet drops to 0.3 Pa. Maintain this vacuum degree and keep it warm for 200 minutes.

[0072] Example 5

[0073] A process for preparing and filling a live vaccine against porcine reproductive and respiratory syndrome virus and freeze-drying, comprising the following steps:

[0074] 1) Antigen gradient cooling and thawing

[0075] Take out the porcine reproductive and respiratory syndrome virus antigen stored at -40°C, put it in a -20°C cold storage for 36 hours, then put it in an 8°C cold storage for 24 hours, and then thaw the antigen on a large-capacity shaker with the shaker temperature set at 25°C. When the antigen is completely thawed and there is no more condensed water on the bottle wall, and the antigen is about 10°C at this time, take out the antigen from the shaker, transfer it to the antigen transfer room, shake the antigen well and transfer it to the seedling preparation tank through a pipeline; start the stirring paddle in the seedling preparation tank to further mix well;

[0076] 2) Seedling preparation

[0077] Adjust the rotation speed of the stirring paddle in the seedling-matching tank to 150 r / min. Inject the protectant kept at 37°C in the protectant preparation tank into the seedling-matching tank through a pipeline while stirring. After the protectant is added to the required dosage, close the valve and continue stirring for 4 min. Adjust the pH value of the suspension to 6.6 and continue stirring for 12 min. Then pump the prepared finished product into the filling system through a pipeline;

[0078] 3) Filling:

[0079] Use a fully automatic filling system for filling;

[0080] 4) Freeze-drying

[0081] 4-1) Pre-freezing:

[0082] Put the filled product into the drying chamber at 26°C. When the temperature of the product drops to 0°C, raise the temperature to 20°C and keep it warm for 60 min. Then continue to cool it to -38°C and keep it warm for 300 min;

[0083] 4-2) Sublimation drying:

[0084] After pre-freezing, evacuate the air. When the vacuum drops to 30 Pa, start heating. The temperature of the shelf is 3°C and the vacuum degree does not exceed 25 Pa. Maintain this vacuum degree and keep it warm for 700 min;

[0085] 4-3) Desorption drying:

[0086] Raise the temperature of the product obtained by sublimation drying to 28°C. The vacuum degree in the chamber drops to 5 Pa. Maintain this vacuum degree and keep it warm for 300 min.

Claims

1. A preparation, filling and freeze-drying process for a live vaccine against porcine reproductive and respiratory syndrome virus, characterized in that, It includes the following steps: 1) Antigen gradient cooling and thawing Take out the porcine reproductive and respiratory syndrome virus antigen stored at -40°C, cool it down and thaw it by gradient, then transfer it to the antigen transfer room. After shaking the antigen evenly, transfer it to the vaccine preparation tank through a pipeline, and start the agitator paddle in the vaccine preparation tank to further mix it evenly; 2) Vaccine preparation Pump the protectant kept at 37°C in the protectant preparation tank into the vaccine preparation tank through a pipeline while stirring. When the protectant is added to the required dosage, close the valve and continue stirring for 3 - 4 minutes. Adjust the pH of the suspension to 6.5 - 6.8, and continue stirring for 10 - 12 minutes until it is uniform. Then pump the prepared finished product into the filling system through a pipeline; 3) Filling: Use a fully automatic filling system for filling; 4) Freeze-drying 4-1) Pre-freezing: Put the filled product into the drying cabinet at 18 - 26°C. When the temperature of the product drops to -4 - 0°C, then raise the temperature to 18 - 20°C and keep it warm for 50 - 60 minutes, and then continue to cool it down to -40 - 38°C and keep it warm for 250 - 300 minutes; 4-2) Sublimation drying: After pre-freezing, evacuate the air. When the vacuum drops to 28 - 30 Pa, start heating. The temperature of the plate layer is -5 - 3°C, and the vacuum degree does not exceed 25 Pa. Maintain this vacuum degree and keep it warm for 400 - 700 minutes; 4-3) Desorption drying: Heat up the product obtained by sublimation drying to 20 - 28°C, and the vacuum degree in the cabinet drops to 0.3 - 5 Pa. Maintain this vacuum degree and keep it warm for 200 - 300 minutes.

2. The seedling preparation, filling and freeze-drying process of a live PRRS virus vaccine according to claim 1, characterized in that, The antigen is cooled and thawed by gradient in the following way: Take out the porcine reproductive and respiratory syndrome virus antigen stored at -40°C, put it into a cold storage at -20°C and store it for 24 - 36 hours, then put it into a cold storage at 2 - 8°C and store it for 15 - 24 hours. Then place the antigen on a shaker to thaw it. The temperature of the shaker is set at 22 - 25°C. When the antigen is completely thawed and there is no more condensed water on the bottle wall, take out the antigen from the shaker.

3. The dispensing, filling and freeze-drying process of a live PRRS virus vaccine according to claim 1, characterized in that, In step 2), the rotation speed of the agitator paddle in the vaccine preparation tank is 120 - 150 r / min.

4. The vaccine preparation, filling and freeze-drying process of a live PRRS virus vaccine according to claim 1, characterized in that, In step 2), adjust the pH of the suspension to 6.6.

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