A method for rapidly screening finished vaccine products using dynamic light scattering technology

Abstract

This application relates to the technical field of vaccine quality detection, and specifically discloses a method for rapidly screening vaccine finished products using dynamic light scattering technology. The method includes the following steps: First, determine the relationship between the particle size of the vaccine and its titer and thermal stability, and establish a dynamic light scattering database; then compare the particle size of the vaccine to be tested with the dynamic light scattering database to screen out the optimal dilution ratio for vaccine synthesis. This application can achieve the rapid screening of vaccine finished products, thereby improving the quality and production rate of vaccine finished products, and has the advantages of simple operation and high screening efficiency.

Description

Technical Field

[0001] This application relates to the technical field of vaccine quality detection, and specifically relates to a method for rapidly screening vaccine products using dynamic light scattering technology. Background Art

[0002] Traditional inactivated virus vaccines are prepared by inoculating viruses onto suitable animal cells. After propagation, the viruses are released into the culture medium. Through clarification, ultrafiltration concentration, inactivation, and purification, the inactivated virus vaccine stock solution is obtained. To enable the inactivated virus to be stored for a longer time, a protective agent needs to be added. The inactivated virus vaccine stock solution and the protective agent are mixed to form the inactivated virus vaccine. Since the vaccine product is composed of the inactivated virus vaccine stock solution and the protective agent, in order to reduce side effects, the inactivated virus vaccine stock solution is diluted during the vaccine synthesis process. To ensure the immunogenicity of the diluted vaccine product, its titer needs to be detected. In addition, a thermal stability accelerated experiment needs to be carried out to detect the shelf life of the vaccine. By combining the two, the effectiveness and stability of the vaccine are determined.

[0003] Currently, animal experiments are generally used for the titer and thermal stability detection of traditional inactivated virus vaccines. However, animal experiments have certain drawbacks: First, affected by different animal batches, the detection results of vaccine products cannot be quantitatively compared, and batch-to-batch differences cannot be reflected. Second, the detection cycle of animal experiments is relatively long, and the effectiveness and stability of the current vaccine at the current dilution ratio cannot be rapidly screened, thus restricting the production rate of vaccines. Summary of the Invention

[0004] In order to rapidly screen the effectiveness and stability of vaccine products and improve the quality and production rate of vaccine products, this application provides a method for rapidly screening vaccine products using dynamic light scattering technology.

[0005] This application provides a method for rapidly screening vaccine products using dynamic light scattering technology, adopting the following technical solutions:

[0006] A method for rapidly screening vaccine products using dynamic light scattering technology includes the following steps:

[0007] (1) Determine the relationship between the particle size of the vaccine and its titer and thermal stability, and establish a dynamic light scattering database;

[0008] (2) Compare the particle size of the vaccine to be tested with the dynamic light scattering database to screen out the optimal dilution ratio for vaccine synthesis.

[0009] The method for rapidly screening vaccine products using dynamic light scattering technology provided by this application can achieve rapid screening of the effectiveness and stability of inactivated virus vaccines, and then optimize the best vaccine synthesis dilution ratio. It can reduce the batch - to - batch differences of vaccine products and the impurity content in each dose of vaccine while not affecting the titer of vaccine products and ensuring vaccine stability, thereby improving production stability and product quality.

[0010] Preferably, step (1) is specifically as follows: Take the inactivated virus vaccine stock solution and dilute and synthesize the vaccine in different proportions; detect the particle size of the vaccine, and at the same time detect the titer and thermal stability of the vaccine; then correlate the particle size detection results of the vaccine under the same dilution ratio with the detection results of titer and thermal stability, thereby establishing a dynamic light scattering database.

[0011] This application first detects the particle size of the synthesized vaccine at different dilution ratios, then separately detects its titer and thermal stability, and correlates the particle size results with the titer and thermal stability detection results, which can establish a dynamic light scattering database. The data in this database is accurate and highly reliable, and can quickly judge the titer and thermal stability of the vaccine product according to the particle size of the vaccine product, so as to quickly screen the vaccine and optimize the best vaccine synthesis dilution ratio, thereby improving production efficiency.

[0012] Preferably, step (2) is specifically as follows: Detect the particle size of the vaccine to be tested, and use the dynamic light scattering database to quickly judge the titer and thermal stability of the vaccine to be tested, thereby screening out the best vaccine synthesis dilution ratio.

[0013] This application can quickly judge the titer and thermal stability of the vaccine to be tested by detecting the particle size of the vaccine to be tested. Therefore, it can quickly screen out the optimal ratio of the vaccine product while ensuring the titer and thermal stability, reduce the batch - to - batch differences in production without affecting the titer and thermal stability of the vaccine, and increase the stability of vaccine production. In addition, by controlling the dilution ratio of the vaccine product, while ensuring the effectiveness and stability of the vaccine, the impurity content in the vaccine is reduced, thereby reducing the side effects of the vaccine and ultimately improving the product quality of the vaccine product.

[0014] Preferably, a vaccine protectant is also added during the synthesis of the vaccine;

[0015] Preferably, the vaccine protectant is human serum albumin.

[0016] Furthermore, the concentration of the human serum albumin is 2% - 2.5%.

[0017] In a specific embodiment, the concentration of the human serum albumin can be 2%, 2.5% or 3%.

[0018] In some specific embodiments, the concentration of the human albumin may also be 2.5%-3%.

[0019] In the present application, adding a vaccine protectant during the vaccine synthesis process can enable the inactivated virus vaccine to exhibit a stable particle size distribution pattern, and the average particle size of the inactivated virus vaccine can be obtained through dynamic light scattering technology. At the same time, the vaccine protectant can also increase the aggregation temperature of the vaccine and play a good protective role for the inactivated virus particles in the vaccine.

[0020] The vaccine contains a large number of inactivated virus particles, and the amount of inactivated virus particles directly affects the potency of the vaccine. Since each inactivated virus particle has its own particle size, affected by the particle size, the stability of the inactivated virus particles is poor. Therefore, adding a protectant during the vaccine synthesis process can enable the inactivated virus particles in the vaccine product to exhibit a stable particle size distribution pattern, and the average particle size of the vaccine product solution can be detected through dynamic light scattering technology.

[0021] Preferably, the vaccine is a Japanese encephalitis inactivated virus vaccine.

[0022] Preferably, the optimal vaccine synthesis dilution ratio of the Japanese encephalitis inactivated virus vaccine is 1:(1-5).

[0023] In a specific embodiment, the optimal vaccine synthesis dilution ratio of the Japanese encephalitis inactivated virus vaccine may be 1:1, 1:2, 1:3, 1:4 or 1:5.

[0024] In some specific embodiments, the optimal vaccine synthesis dilution ratio of the Japanese encephalitis inactivated virus vaccine may also be 1:(1-2), 1:(1-3), 1:(1-4), 1:(2-3), 1:(2-4), 1:(2-5), 1:(3-4), 1:(3-5) or 1:(4-5).

[0025] In the present application, the Japanese encephalitis inactivated virus vaccine is screened by the method of rapidly screening vaccine products using dynamic light scattering technology, and the optimal vaccine synthesis dilution ratio of the obtained Japanese encephalitis inactivated virus vaccine is 1:(1-5). The vaccine obtained under this vaccine synthesis dilution ratio has good potency and thermal stability and low impurity content.

[0026] Preferably, the detection time of the method of rapidly screening vaccine products using dynamic light scattering technology is less than 10 h.

[0027] The method of rapidly screening vaccine products using dynamic light scattering technology provided by the present application can detect the effectiveness and stability of the vaccine product at a certain dilution ratio within 10 h, greatly improving the production efficiency of the vaccine. This method has the characteristics of simple operation, good stability of detection results and short detection cycle.

[0028] In summary, the present application has the following beneficial effects:

[0029] 1. The method for rapidly screening vaccine finished products using dynamic light scattering technology provided by the present application can achieve rapid screening of the effectiveness and stability of inactivated virus vaccines, and then optimize the best synthesis dilution ratio of the vaccine. It can reduce the batch - to - batch differences of vaccine finished products and the impurity content in each dose of vaccine on the premise of not affecting the titer of vaccine finished products and ensuring vaccine stability during production, thereby improving production stability and product quality.

[0030] 2. In the present application, adding a vaccine protectant during the vaccine synthesis process can make the inactivated virus vaccine present a stable particle size distribution pattern, and the average particle size of the inactivated virus vaccine can be obtained through dynamic light scattering technology. At the same time, the vaccine protectant can also increase the aggregation temperature of the vaccine and play a good protective role for the inactivated virus particles in the vaccine.

[0031] 3. The method for rapidly screening vaccine finished products using dynamic light scattering technology provided by the present application can detect the effectiveness and stability of the finished vaccine at a certain dilution ratio within 10 h, greatly improving the production efficiency of the vaccine. This method has the characteristics of simple operation, good stability of detection results, and short detection cycle. Specific Embodiments

[0032] The present application provides a method for rapidly screening vaccine finished products using dynamic light scattering technology, which includes the following steps:

[0033] (1) First, dissolve human serum albumin with PBS solution to obtain a dilution solution; then take the inactivated virus vaccine stock solution and dilute it with the dilution solution according to different ratios to synthesize the inactivated virus vaccine; detect the particle size of the inactivated virus vaccine, and at the same time detect the titer and thermal stability of the vaccine; correlate the particle size detection results of the inactivated virus vaccine at the same dilution ratio with the detection results of titer and thermal stability, so as to establish a dynamic light scattering database. Among them, in the inactivated virus vaccine, the concentration of human serum albumin is 2% - 2.5%.

[0034] (2) Detect the particle size of the in - test inactivated virus vaccine. The dynamic light scattering database can quickly judge the titer and thermal stability of the inactivated virus vaccine according to the particle size of the in - test inactivated virus vaccine, so as to screen out the best synthesis dilution ratio of the inactivated virus vaccine.

[0035] The vaccine used in the present application is Japanese encephalitis inactivated virus vaccine.

[0036] The human serum albumin used in the present application is purchased from Hualan Biological Engineering Chongqing Co., Ltd.; the rest of the reagents, solvents, etc. can all be obtained through commercial purchase.

[0037] The following further elaborates on the present application in combination with preparation examples and implementation examples.

[0038] Preparation Example

[0039] Preparation Examples 1 - 7

[0040] Preparation Examples 1 - 7 provide a Japanese encephalitis inactivated virus vaccine, using the following method:

[0041] The difference in the above - mentioned preparation examples lies in: during the preparation process of the Japanese encephalitis inactivated virus vaccine, the dilution ratio of the Japanese encephalitis inactivated virus vaccine stock solution is specifically shown in Table 1.

[0042] The preparation method of the Japanese encephalitis inactivated virus vaccine is as follows: First, weigh human serum albumin according to a ratio, dissolve it in PBS solution to obtain a dilution solution, and control the salt concentration in the dilution solution to be 0.145 mM and the pH to be 7 - 8; then take 10 mg of the Japanese encephalitis inactivated virus vaccine stock solution and mix it with the above - mentioned dilution solution to obtain the Japanese encephalitis inactivated virus vaccine. Among them, the concentration of human serum albumin in the Japanese encephalitis inactivated virus vaccine is 2%.

[0043] Table 1 Dilution ratio of the Japanese encephalitis inactivated virus vaccine stock solution in Preparation Examples 1 - 7

[0044]

[0045] Preparation Examples 8 - 10

[0046] Preparation Examples 8 - 10 provide a Japanese encephalitis inactivated virus vaccine, using the following method:

[0047] The difference between the above - mentioned preparation examples and Preparation Example 3 lies in: during the preparation process of the Japanese encephalitis inactivated virus vaccine, the concentration of human serum albumin is specifically shown in Table 2.

[0048] Table 2 Concentrations of human serum albumin in the Japanese encephalitis inactivated virus vaccines provided by Preparation Example 3 and Preparation Examples 8 - 10

[0049]

[0050]

[0051] Test Results

[0052] The Japanese encephalitis inactivated virus vaccines provided by Preparation Examples 1 - 10 were tested, and their particle sizes, aggregation temperature T agg value, titer, and thermal stability were measured respectively, and the test results were compared.

[0053] Particle Size and Aggregation Temperature T agg value (the starting temperature at which proteins form aggregates) was detected using a WYATT DynaProPlate Reader III high - throughput protein solution stability analyzer (DLS); the titer and thermal stability were detected using pharmacopoeia standards.

[0054] Potency is an important indicator for detecting vaccines and measures whether the vaccine can produce antibodies in the organism. The current potency detection method is to detect the sample and the standard simultaneously, and then take the difference of the detection results. If the difference is positive, it indicates that the ability of the vaccine to produce antibodies in the organism is not inferior to that of the standard, and the vaccine is effective; if the difference is negative, it indicates that the ability of the vaccine to produce antibodies in the organism is insufficient, and the vaccine is ineffective.

[0055] For the thermal stability test, the potency is detected after placing at 37 °C for 1 week. If the potency is qualified, it indicates that the vaccine has good stability and can meet the requirements within the shelf life; if it is unqualified, it indicates that the vaccine has insufficient stability and cannot guarantee to be qualified within the shelf life.

[0056] Table 3 Detection results of the inactivated JE virus vaccine stock solution and the inactivated JE virus vaccines provided in Preparation Examples 1-10

[0057]

[0058]

[0059] Combined with Table 1 and Table 3 and according to the detection results of Preparation Examples 1-7, it can be seen that as the dilution ratio of the inactivated JE virus vaccine stock solution increases, the average particle size of the inactivated JE virus vaccine shows a decreasing trend, the aggregation temperature T agg value remains stable all the time, and there is no obvious rule for the potency and thermal stability. Further comparison shows that the potencies of the inactivated JE virus vaccines with a dilution ratio of 1:(1-5) provided in Preparation Examples 1-5 are all positive, and the particle sizes of the corresponding inactivated JE virus vaccines are ≥16 nm, while the potencies of the inactivated JE virus vaccines with a dilution ratio of 1:(6-7) provided in Preparation Examples 6-7 are all negative, and the particle sizes of the corresponding inactivated JE virus vaccines are <16 nm. Therefore, to ensure the qualified potency and thermal stability of the vaccine, the particle size of the inactivated JE virus vaccine should be ≥16 nm, and the dilution ratio of the inactivated JE virus vaccine stock solution should be controlled within the range of 1:(1-5).

[0060] According to the detection results of Preparation Examples 3 and Preparation Examples 8-10, it can be seen that as the concentration of human serum albumin in the inactivated JE virus vaccine increases, the particle size of the inactivated JE virus vaccine shows a gradually decreasing trend, and the aggregation temperature T agg value gradually increases, indicating that human serum albumin can increase the aggregation temperature of the inactivated JE virus vaccine and enhance the protection effect on the inactivated JE virus vaccine. Therefore, in order to reduce the synthesis cost of the vaccine, when the concentration of human serum albumin in the inactivated JE virus vaccine is controlled within the range of 2%-2.5%, the protective effect of human serum albumin on the vaccine is the best, and the prepared vaccine has good thermal stability.

[0061] By comparing the detection of the particle size and the aggregation temperature T using a stability analyzer aggThe time for measuring the value and the time for detecting the potency and thermal stability using pharmacopoeia standards were compared, and it was found that the time for detecting the particle size and the aggregation temperature T using a stability analyzer was 8 h; the time for detecting the potency and thermal stability using pharmacopoeia standards was 55 days. Therefore, the method for rapidly screening finished vaccines provided by this application has the advantages of simple operation method, low detection cost, and fast screening speed. agg The time for measuring the value and the time for detecting the potency and thermal stability using pharmacopoeia standards were compared, and it was found that the time for detecting the particle size and the aggregation temperature T using a stability analyzer was 8 h; the time for detecting the potency and thermal stability using pharmacopoeia standards was 55 days. Therefore, the method for rapidly screening finished vaccines provided by this application has the advantages of simple operation method, low detection cost, and fast screening speed.

[0062] Accelerated thermal stability test

[0063] The inactivated JE virus vaccine provided in Preparation Example 3 was placed at 25°C and 37°C respectively for accelerated thermal stability study, and the particle size and potency of the samples were detected weekly. The test results are shown in Table 4.

[0064] Table 4 Results of accelerated thermal stability test of the inactivated JE virus vaccine provided in Preparation Example 3

[0065]

[0066] From the above results of the thermal stability test, it was found that when the inactivated JE virus vaccine was stored at 25°C and 37°C respectively, with the extension of the storage time, the particle size and potency of the inactivated JE virus vaccine both showed a downward trend, indicating that storing the inactivated JE virus vaccine at high temperature would reduce its particle size and potency, and the higher the temperature, the faster the decrease. Further comparison found that when the particle size of the inactivated JE virus vaccine < 16 nm, the potency of the inactivated JE virus vaccine showed a negative value. Therefore, to ensure the potency of the vaccine, the particle size of the inactivated JE virus vaccine should be controlled ≥ 16 nm.

[0067] Long-term stability test

[0068] The inactivated JE virus vaccine provided in Preparation Example 3 was stored at 2 - 8°C respectively, and the particle size and potency were detected every 3 months. After 18 months, the particle size and potency were detected every 1 month. The test results are shown in Table 5.

[0069] Table 5 Results of long-term stability test of the inactivated JE virus vaccine provided in Preparation Example 3

[0070]

[0071] From the above results of the long-term stability test, it was found that when the inactivated JE virus vaccine was stored at 5°C, with the extension of the storage time, the particle size and potency of the inactivated JE virus vaccine both showed a downward trend. Further comparison found that under the insulation condition of 5°C, when the storage time of the inactivated JE virus vaccine exceeded 18 months, the potency of the inactivated JE virus vaccine became negative, and at this time the particle size of the inactivated JE virus vaccine < 16 nm. Therefore, to ensure the potency of the vaccine, the particle size of the inactivated JE virus vaccine should be controlled ≥ 16 nm.

[0072] Test detection results

[0073] From the detection results of Preparation Examples 1-10, accelerated thermal stability test and long-term stability test, it can be seen that there is a direct relationship between the particle size of the inactivated JE virus vaccine and its titer and thermal stability, and a corresponding dynamic light scattering database has been established. In the dynamic light scattering database, the particle size of the corresponding inactivated JE virus vaccine corresponds to the corresponding titer. To ensure the effectiveness of the vaccine, its titer should be positive, and the particle size of the corresponding inactivated JE virus vaccine should be ≥16 nm. Therefore, when using the method of quickly screening the finished vaccine by dynamic light scattering technology in this application to detect the vaccine, only the particle size of the vaccine needs to be detected, and then the titer and thermal stability of the vaccine can be known according to the obtained dynamic light scattering database, so as to quickly screen out the best dilution ratio of the vaccine stock solution and ensure the effectiveness of the vaccine at the same time.

[0074] Reliability verification

[0075] Take 10 batches of inactivated JE virus vaccine samples, detect the particle size of the samples respectively, and obtain the titer and thermal stability of the 10 batches of vaccines under the above particle size according to the dynamic light scattering database; at the same time, use the pharmacopoeia standard to detect the titer and thermal stability; compare the titer and thermal stability detection results obtained by the two methods respectively to verify the reliability of the dynamic light scattering database. The detection results are shown in Table 6.

[0076] Table 6 Reliability verification results

[0077]

[0078] From the above reliability verification results, it is found that the method provided in this application for quickly screening the finished vaccine by dynamic light scattering technology can obtain its titer and thermal stability through the particle size of the inactivated JE virus vaccine, and the titer and thermal stability results obtained are basically the same as those obtained by the pharmacopoeia standard detection, indicating that the data in the dynamic light scattering database is reliable.

[0079] Examples

[0080] Example 1

[0081] Example 1 provides a method for quickly screening the finished vaccine by dynamic light scattering technology.

[0082] The above method includes the following steps:

[0083] (1) According to the detection results of Preparation Examples 1-10, accelerated thermal stability test and long-term stability test, correlate the particle size detection results of the inactivated JE virus vaccine with the titer and thermal stability detection results, and establish a dynamic light scattering database.

[0084] (2) Select the inactivated JE virus vaccine products synthesized at different dilution ratios under the same batch, and use the WYATT DynaPro Plate Reader III high-throughput protein solution stability analyzer to detect the particle sizes of each inactivated JE virus vaccine product; use the dynamic light scattering database to quickly judge the potency and thermal stability of the inactivated JE virus vaccine at each particle size, and at the same time detect the impurity content (residual Vero cell protein) in each inactivated JE virus vaccine, so as to screen out the optimal synthesis dilution ratio of the inactivated JE virus vaccine.

[0085] The residual Vero cell protein is determined by the enzyme-linked immunosorbent assay (General Rule 3429) in the pharmacopoeia standard. According to the national standard, the residual Vero cell protein in the vaccine product should not be higher than 1 μg / dose. Calculated according to 0.5 ml per dose, that is, not higher than 2 μg / ml.

[0086] The dilution ratio, particle size, potency, thermal stability and impurity content of the inactivated JE virus vaccine products are shown in Table 7.

[0087] Table 7 Relevant data of the inactivated JE virus vaccine products in the method provided in Example 1

[0088]

[0089] It can be seen from the above table that the method for quickly screening vaccine products using dynamic light scattering technology provided by this application can complete the particle size detection of inactivated JE virus vaccine products and obtain the results of potency and thermal stability within 10 h. Through comparison, it is found that as the dilution ratio increases, the particle size and impurity content of the inactivated JE virus vaccine products gradually decrease, and there is still no obvious rule for the potency and thermal stability of the inactivated JE virus vaccine products; further comparison shows that when the dilution ratio of the inactivated JE virus vaccine product is 1:5, the average particle size of the inactivated JE virus vaccine product is close to 16 nm, its potency is qualified, but its thermal stability is unqualified; when the dilution ratio of the inactivated JE virus vaccine product is 1:(1 - 3), the residual Vero cell protein in the inactivated JE virus vaccine product > 2 μg / ml, which does not meet the national standard; while when the dilution ratio of the inactivated JE virus vaccine product is 1:4, the potency and stability of the inactivated JE virus vaccine product are qualified, and the residual Vero cell protein is small. Therefore, by screening the inactivated JE virus vaccine products using the method for quickly screening vaccine products using dynamic light scattering technology provided by this application, the optimal synthesis dilution ratio of the inactivated JE virus vaccine is obtained as 1:4.

[0090] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for rapidly screening finished vaccine products using dynamic light scattering technology, characterized in that, it includes the following steps: (1) Take the inactivated virus vaccine stock solution and dilute and synthesize the vaccine according to different ratios; detect the particle size of the vaccine, and at the same time detect the potency and thermal stability of the vaccine; then correlate the particle size detection results of the vaccine under the same dilution ratio with the detection results of potency and thermal stability, so as to establish a dynamic light scattering database; (2) Detect the particle size of the vaccine to be tested, and use the dynamic light scattering database to quickly judge the potency and thermal stability of the vaccine to be tested. When the potency of the vaccine to be tested is positive and the residual amount of Vero cell protein ≤ 2 μg / mL, the dilution ratio of the vaccine to be tested is the optimal vaccine synthesis dilution ratio; The detection time of the method for rapidly screening finished vaccine products using dynamic light scattering technology is less than 10 h.

2. The method for rapidly screening finished vaccine products using dynamic light scattering technology according to claim 1, characterized in that, a vaccine protectant is also added during the synthesis of the vaccine.

3. The method for rapidly screening finished vaccine products using dynamic light scattering technology according to claim 2, characterized in that, the vaccine protectant is human serum albumin.

4. The method for rapidly screening finished vaccine products using dynamic light scattering technology according to claim 3, characterized in that, the concentration of the human serum albumin is 2% - 2.5%.

5. The method for rapidly screening finished vaccine products using dynamic light scattering technology according to claim 1, characterized in that, the vaccine is an inactivated Japanese encephalitis virus vaccine.

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