Mass production and inactivation process for foot and mouth disease virus

Abstract

The invention discloses a large-scale production and inactivation process for foot and mouth disease viruses. After standardization of an inactivator cyclization method is established, storage time and temperature of the inactivator are optimized, toxicity test of 50% Na2S2O3 on BHK-21 cells and dosage optimization of inactivated 50% Na2S2O3 in mass production are carried out, and different foot-and-mouth disease virus strains TCID50 and inactivation kinetics curves are established, the method is applied to large-scale production. After cyclization of the inactivator, whether BEA cyclization succeeds or not is judged by detecting pH and color change of a beta-naphthol purple indicator, thorough inactivation of venom is further ensured, biological safety is improved, meanwhile, it is determined that the inactivator can be stored at the temperature of 2-8 DEG C, and the storage life is smaller than or equal to 14 d; 50% of Na2S2O3 is not added after venom inactivation is finished, so that side reactions after vaccination are reduced, the safety of vaccination is improved, and the production cost of the vaccine is reduced; the inactivation process can be accurately controlled, the inactivation efficiency can be improved, the complete inactivation of viruses can be ensured, the biological safety risk in vaccine production can be avoided, the inactivation time can be shortened, and the production cycle can be shortened by drawing the inactivation power curve of different strains.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biological products, and in particular to a large-scale production and inactivation process of foot-and-mouth disease virus. Background Art

[0002] Foot-and-mouth disease is an acute, highly contagious animal disease caused by the foot-and-mouth disease virus (FMDV), which mainly infects even-toed ungulates such as pigs, cattle, and sheep. The disease spreads quickly and widely. Vaccination is the main means of controlling foot-and-mouth disease, and many foot-and-mouth disease outbreaks are related to incomplete inactivation of the foot-and-mouth disease vaccine virus. "Research on Optimization of Inactivation Process of Suspension Culture Foot-and-Mouth Disease Virus" 2021, Issue 06, Page 22. At present, the production of foot-and-mouth disease vaccine mainly depends on the inactivation process of the virus. Under normal circumstances, the live virus content of foot-and-mouth disease virus becomes lower and lower during the inactivation process, and the detection of live virus becomes more and more difficult. Traditional inactivation technology has many shortcomings, which limits the large-scale and efficient production and application of vaccines. The following deficiencies exist in the inactivation process in the large-scale production of foot-and-mouth disease virus: (1) There is no definitive method to determine whether the BEA (dibromoethylamine hydrobromide) cyclization is successful: Literature reports that the optimal pH for inactivating foot-and-mouth disease virus with BEI is 7.5-7.6. During the initial culture process, a phosphate buffer system is used throughout, and the pH at virus harvest is likely to be higher than 7.8. To better control the pH of the venom, the pH of the BEI needs to be controlled. At low pH, BEI may convert to BEA, so an indicator must be added when preparing low-pH BEI to verify the success of the cyclization process. Low pH conditions also shorten the storage time of BEI, so the storage time of BEI should be shortened when using low-pH BEI. Furthermore, when preparing the β-naphthol violet solution, only the color of the inactivator after adding the indicator is described, without explaining the color change or the operation process. ("Study on Optimization of Inactivation Process for Foot-and-Mouth Disease Virus in Suspension Culture," 2021, Issue 06, Pages 23, 29). The above literature emphasizes testing the pH after the inactivator is cyclized during production. This single test indicator is not used simultaneously with pH testing and indicator addition in production. Therefore, it is impossible to determine the success of BEA cyclization in large-scale production. A review of the literature related to foot-and-mouth disease revealed almost no research on the formulation, storage time and temperature of inactivating agents.

[0003] (II) Traditional inactivation time is long in large-scale production: 1. Traditional inactivation time in large-scale production: The suspension culture of foot-and-mouth disease virus was inactivated twice at 30°C with BEI at a concentration of 0.1 mol / L and a final concentration of 3%, each time for 24 hours, for a total of 48 hours. The inactivation process was reliable, the inactivation results were safe, and met the relevant standards. "Study on the Inactivation Technology of Foot-and-mouth Disease Virus in Suspension Culture" 2021, Issue 06, Page 31. The best method for inactivating foot-and-mouth disease virus is to use 2mmoL / L BEI at 30°C for 28 hours. This method can completely inactivate foot-and-mouth disease virus produced by suspension culture and has good safety. "Chinese Journal of Veterinary Drugs" 2012, 46 (5), Page 39. The above literature shows that the effective inactivation time of foot-and-mouth disease virus is 28h or 48h, but none of them indicates how the specific time is determined, nor does it indicate the effective inactivation time of different strains of foot-and-mouth disease virus. By studying the effective inactivation time of different strains of foot-and-mouth disease virus, on the one hand, we can shorten the production cycle and reduce 146S losses; on the other hand, we can save energy, reduce vaccine production costs and improve economic benefits.

[0004] 2. TCID of different strains of foot-and-mouth disease venom 50 And draw the inactivation kinetics curve: During the inactivation process, BEI was added twice with a final concentration of 3mmoL / L at 30°C for a total of 48 hours. The venom was usually taken from the first 4 hours to measure TCID 50 And draw the inactivation force trend graph to confirm the effectiveness of the inactivation process. From "Study on the Inactivation Process of Foot-and-Mouth Disease Virus in Suspension Culture Production" 2021, Issue 06, Page 27. The above literature has a long inactivation time, resulting in a long production cycle and increased 146S loss, and sampling and TCID measurement during the inactivation process 50 It takes at least 3-4 days to determine the inactivation time of FMD venom, while the inactivation time is 48 hours. 50 The method for drawing the inactivation kinetic curve is not designed properly, and the TCID of different strains is tested in large-scale production. 50 Draw the inactivation activity curve, retest the inactivation activity curves of different strains every few months, optimize the inactivation process parameters, and determine how much time is required to completely inactivate different strains of foot-and-mouth disease virus during the inactivation process.

[0005] (III) Optimization of the dosage of 50% Na2S2O3 after inactivation in large-scale production: After inactivation is complete, 2% by volume and 50% Na2S2O3 are added to block and neutralize the remaining inactivator. (Source: "Study on Inactivation Technology of Foot-and-Mouth Disease Virus in Suspension Culture Production," 2021, Issue 6, Page 23.) The above literature does not clearly indicate how to determine whether 2% of 50% Na2S2O3 can neutralize the residual inactivator. There is a lack of research on the dosage of 50% Na2S2O3, and there is no corresponding theoretical data on whether adding 2% of 50% Na2S2O3 can completely neutralize the residual inactivator.

[0006] The present invention solves the shortcomings of the inactivation method in large-scale production of foot-and-mouth disease virus by optimizing the following process: (1) Standardization of Inactivator Formula and Cyclization Method: This patent uses the inactivator formula in the veterinary pharmacopoeia, reducing the proportion of NaOH. β-Naphthol Violet is then added to the prepared solution and heated for cyclization. Successful BEA cyclization is determined by measuring the pH and color change of the β-Naphthol Violet indicator after BEA cyclization. Successful cyclization is indicated by a gradual change from the initial purple color to a light orange color. Combined with pH testing, this ensures complete venom inactivation and enhances biosafety. After inactivating the venom with different inactivator formulas, the venom is then subjected to a temperature challenge for 4 days, and the 146S concentration (μg / ml) is measured. The formulation with the highest 146S stability is selected as the preferred formulation.

[0007] (II) Optimizing the storage time and temperature of inactivators: Inactivators must be stored under specific temperature and time conditions to ensure they can effectively inactivate viruses without compromising their antigenicity and immunogenicity. Inappropriate temperature conditions may cause the inactivator to degrade, affecting its chemical properties and inactivation effectiveness. Appropriate storage conditions can extend the inactivator's lifespan and ensure optimal inactivation effectiveness. Therefore, the correct storage time and temperature are crucial for the effective use of inactivators.

[0008] (III) Toxicity test of 50% Na2S2O3 on BHK-21 cells and optimization of the dosage of 50% Na2S2O3 after inactivation in large-scale production: 1. Toxicity test of 50% Na2S2O3 on BHK-21 cells: As the amount of 50% Na2S2O3 is reduced, the cytotoxicity decreases, and it has no effect on the subsequent antigen purification, emulsification, and packaging.

[0009] 2. Optimizing the addition of 50% Na2S2O3 after inactivation in large-scale production: The efficacy, safety, and physical properties of the vaccine produced after process conversion are consistent with those of the vaccine containing 50% Na2S2O3. By eliminating the addition of 50% Na2S2O3, vaccine stability can be improved, degradation during storage and transportation can be reduced, and the shelf life of the vaccine can be extended. It can also reduce side effects after vaccination and improve vaccination safety. It can help improve the immunogenicity of the vaccine and enhance the immune response of vaccinated animals, thereby improving the protective effect of the vaccine. It can also avoid unnecessary waste and reduce vaccine production costs. It can also reduce the use of chemical reagents and reduce the impact on the environment and animals.

[0010] (IV) TCID of different strains of foot-and-mouth disease virus 50 Study on establishing inactivation kinetics curve: Foot-and-mouth disease virus TCID 50 Tissue Culture Infective Dose 50 (TCI50) is a method for measuring viral titer. It represents the amount of virus capable of infecting 50% of host cells under specific conditions. It can be used to estimate the strength of viral infectivity and the amount of virus present. Relevant patent documents retrieved from the prior art indicate that the inactivation kinetics curve is a mathematical model that describes the change in viral concentration over time during the viral inactivation process. By establishing an inactivation kinetics curve, the inactivation process can be precisely controlled, inactivation conditions can be optimized, inactivation efficiency can be improved, and complete viral inactivation can be ensured, thereby minimizing biosafety risks in vaccine production. It can also shorten inactivation time, reduce production cycle time, minimize 146S antigen loss, improve production efficiency, and reduce vaccine production costs.

[0011] In summary, based on the shortcomings of the existing inactivation technology, the present invention has established a standardized inactivator cyclization method, optimized the storage time and temperature of the inactivator, conducted a toxicity test of 50% Na2S2O3 on BHK-21 cells and optimized the dosage of 50% Na2S2O3 after inactivation in large-scale production, and optimized the TCID of different strains of foot-and-mouth disease virus. 50 The invention improves the inactivation performance of the inactivator, maintains better stability during the use period of the inactivator, and shortens the inactivation time. It has important practical significance for improving vaccine production efficiency, reducing production costs, and improving immune efficacy, and provides a new technical path for the large-scale production of foot-and-mouth disease vaccines. Summary of the Invention

[0012] The purpose of the present invention is to provide a large-scale production and inactivation process for foot-and-mouth disease virus in response to the deficiencies of the current existing technology.

[0013] A large-scale production inactivation process for foot-and-mouth disease virus comprises the following steps: (1) Preparation of the inactivator and establishment of the cyclization method: Dissolve dibromoethylamine hydrobromide (BEA) in injection water, add NaOH solution, adjust the ratio of NaOH to BEA, add β-naphthol violet indicator, detect pH value and color change, and judge whether BEA cyclization is successful. When the color changes from purple to light orange and the pH is between 7.7-7.9, it means that the inactivator cyclization is successful. Store at 2-8℃ and the shelf life is ≤14 days. Prepare NaOH and BEA at a ratio of 70g:205g per 10,000 mL. Maintain BEA at 37°C for 10 minutes to ensure successful cyclization and enhance biosafety. The concentration of the β-naphthol violet indicator solution is 1%. The inactivator should be stored at 4°C for ≤14 days to ensure effective inactivation and antigen stability.

[0014] (2) Venom inactivation: Use the BHK-21 cell suspension method to culture foot-and-mouth disease virus to obtain a mature foot-and-mouth disease virus suspension culture medium; after centrifugation, the virus suspension culture medium is added with a prepared inactivator and inactivated for 28 hours. During the inactivation process, there is no need to add 50% Na2S2O3 to block it, and it can be directly used for subsequent antigen purification production; (3) Plotting the inactivation kinetics curves of different FMDV strains: Add the prepared inactivator to the venom of O / Mya98, AF / 72, and O / MYA98 / BY, add 50% Na2S2O3 to block, and measure the TCID at 0, 1, 2, and 3 h of inactivation. 50 The values ​​are expressed with time as the horizontal axis, and the TCID values ​​of O / Mya98, AF / 72, and O / MYA98 / BY strains at 0, 1, 2, and 3 h of inactivation are plotted. 50 The values ​​were used as the vertical coordinates, and the inactivation activity curve was drawn to optimize the inactivation process parameters. It was found that the inactivation time of the AF / 72 strain was 15.99-16.47 h, the inactivation time of the O / MYA98 / BY strain was 22.96-24.38 h, and the inactivation time of the O / Mya98 strain was 18.86-18.94 h.

[0015] After the inactivator formula of the present invention is formulated according to the ratio of the inactivator formula in the veterinary pharmacopoeia, the pH of BEI is higher than the pH of the venom in large-scale production. In order to make the pH of the two closer and facilitate subsequent production, the proportion of NaOH is reduced in the present invention to optimize the inactivator formula; traditional methods cannot determine the progress of the BEA cyclization reaction in real time, resulting in uncontrollable inactivator activity. By detecting the pH and adding the color change of the β-naphthol violet indicator to determine whether the BEA cyclization is successful, it is further ensured that the venom is completely inactivated and biosafety is improved. In the process of optimizing the inactivator formula and standardizing the cyclization method, the ratio of NaOH to BEA is adjusted, the β-naphthol violet indicator is added, and the pH value and color change are detected to determine whether the BEA cyclization is successful; NaOH and BEA are prepared according to the ratios of 80g:205g, 75g:205g, 70g:205g, and 65g:205g per 10,000 milliliters, the pH value and color change are detected, and the optimal formula is determined to be NaOH and BEA at a ratio of 70g:205g per 10,000 milliliters.

[0016] The storage time and temperature of the inactivator of the present invention are optimized. There is a lack of systematic research on the storage stability of BEI solution. The existing storage conditions cannot determine whether the solution has degraded and lost efficacy. The present invention can improve the inactivation performance of the inactivator and maintain better stability during the use period. The storage period of the inactivator at 4°C is determined to be ≤14 days to ensure the inactivation effect and antigen stability. The inactivator is stored under different temperatures (4°C, 25°C, 37°C) and time (2d, 6d, 10d, 14d, 30d) and the TCID 50 Determine the optimal storage conditions.

[0017] The present invention conducts a toxicity test on BHK-21 cells with 50% Na2S2O3 and optimizes the amount of 50% Na2S2O3 added after inactivation in large-scale production, thereby reducing the amount of 50% Na2S2O3 added, reducing cytotoxicity, and improving vaccine stability and safety. Through a toxicity test on BHK-21 cells with 50% Na2S2O3, it is determined that the amount of 50% Na2S2O3 added is between 0% and 0.2%, and the cell viability is ≥100%. In large-scale production, after inactivation for 28 hours, there is no need to add 50% Na2S2O3, and the vaccine can be directly used in subsequent antigen purification production.

[0018] TCID of different strains of foot-and-mouth disease virus of the present invention (AF / 72 strain, O / MYA98 / BY strain, O / Mya98 strain) 50 Compared with the research method of establishing inactivation kinetic curve, the inactivation kinetics of different virus strains vary significantly, and conventional inactivation process parameters lack scientific quantitative basis. Precisely control the inactivation process, improve inactivation efficiency, ensure complete inactivation of the virus, and avoid biosafety risks in vaccine production. It can also shorten the inactivation time, reduce the production cycle, reduce the loss of antigen 146S, improve production efficiency, and reduce vaccine production costs. By measuring the TCID of different strains50 The inactivation kinetics curve was drawn to optimize the inactivation process parameters and shorten the inactivation time. 50 By using the inactivation kinetics curve, we can optimize the inactivation process parameters, shorten the inactivation time and improve the vaccine production efficiency.

[0019] In summary, the present invention has established the standardization of the inactivator cyclization method, optimized the storage time and temperature of the inactivator, conducted the toxicity test of 50% Na2S2O3 on BHK-21 cells and optimized the dosage of 50% Na2S2O3 after inactivation in large-scale production, and optimized the TCID of different strains of foot-and-mouth disease virus. 50 After the inactivation kinetic curve is established, it is applied in large-scale production. After the inactivator is cyclized, the pH and color change of the β-naphthol violet indicator are tested to determine whether the BEA cyclization is successful, further ensuring that the venom is completely inactivated and improving biosafety. At the same time, it is determined that the inactivator can be stored at 2-8°C with a shelf life of ≤14d; 50% Na2S2O3 is not added after the venom inactivation is completed, which reduces side effects after vaccination, improves the safety of vaccination, and reduces the production cost of the vaccine; by drawing inactivation kinetic curves of different strains, the inactivation process can be accurately controlled, the inactivation efficiency can be improved, the virus can be ensured to be completely inactivated, and biosafety risks in vaccine production can be avoided. It can also shorten the inactivation time and reduce the production cycle.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adds β-naphthol violet solution and reacts with uncyclized BEA to produce a specific color reaction (purple turns light orange), thereby clarifying the BEA cyclization time and process, achieving visual and precise monitoring of the BEA cyclization reaction process, and intuitively judging the optimal cyclization completion time, avoiding errors caused by relying on empirical endpoint speculation, reducing the waste of process raw materials, and increasing the stability and safety of the antigen process.

[0021] (2) Optimize the storage time and temperature of the inactivator to ensure that it can effectively inactivate the virus without damaging its antigenicity and immunogenicity; improper temperature conditions may cause the degradation of the inactivator, affecting its chemical properties and inactivation effect; appropriate storage conditions can extend the use period of the inactivator and ensure that the inactivation effect meets EU requirements when used. Therefore, the correct storage time and storage temperature are crucial for the effective use of the inactivator.

[0022] (3) The present invention measures the TCID of different strains of foot-and-mouth disease virus 50By drawing the inactivation activity curve and establishing an accurate inactivation activity model, compared with the traditional fixed 28-hour inactivation procedure, the inactivation time is shortened, which has important practical significance for improving vaccine production efficiency, reducing production costs and improving immune efficacy. It comprehensively achieves three technological breakthroughs in precise inactivation, energy consumption optimization and product protection, and provides a new technical path for the large-scale production of foot-and-mouth disease vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 for different ratios of inactivator (250mL); Figure 2 1% β-naphthol violet (150 mL) was added to the inactivator at different ratios; Figure 3 Cyclization was performed in different ratios of inactivator for 10 min (150 mL); Figure 4 Cyclization was performed in different ratios of inactivator for 60 min (50 mL); Figure 5 3h TCID after 2 days of inactivation by different temperature inactivators 50 value / 0.1mL (O / MYA98 / BY virus); Figure 6 3h TCID after 6 days of inactivation by different temperature inactivators 50 value / 0.1mL (O / MYA98 / BY virus); Figure 7 3h TCID after 10 days of inactivation by different temperature inactivators 50 value / 0.1mL (O / MYA98 / BY virus); Figure 8 3h TCID after 14 days of inactivation at different temperatures 50 value / 0.1mL (O / MYA98 / BY virus); Figure 9 3hTCID after 30d inactivation by different temperature inactivators 50 value / 0.1mL (O / MYA98 / BY virus); Figure 10 The inactivation curve of AF / 72 strain venom (TCID 50 average value); Figure 11 The inactivation curve of O / MYA98 / BY strain venom (TCID 50 average value); Figure 12 The inactivation curve of O / Mya98 strain venom (TCID 50 average value); Figure 13 The inactivation curve of AF / 72 strain venom (TCID 50 Average value) (retested 2 months later); Figure 14 The inactivation curve of O / MYA98 / BY strain venom (TCID 50 Average value) (retested 2 months later); Figure 15 The inactivation curve of O / Mya98 strain venom (TCID 50 Average value) (retested after 2 months). DETAILED DESCRIPTION

[0024] The foot-and-mouth disease virus inactivation process of the present invention is described in detail below through specific implementation. Example

[0025] The foot-and-mouth disease virus inactivation process of the present invention comprises the following steps: (1) Preparation of the inactivator and establishment of the cyclization method: Dissolve dibromoethylamine hydrobromide (BEA) in injection water, add NaOH solution, adjust the ratio of NaOH to BEA, add β-naphthol violet indicator, detect pH value and color change, and judge whether BEA cyclization is successful. When the color changes from purple to light orange and the pH is between 7.7-7.9, it means that the inactivator cyclization is successful. Store at 2-8℃ and the shelf life is ≤14 days. Prepare NaOH and BEA at a ratio of 70g:205g per 10,000 mL. Maintain BEA at 37°C for 10 minutes to ensure successful cyclization and enhance biosafety. The concentration of the β-naphthol violet indicator solution is 1%. The inactivator should be stored at 4°C for ≤14 days to ensure effective inactivation and antigen stability.

[0026] (2) Venom inactivation: Use the BHK-21 cell suspension method to culture foot-and-mouth disease virus to obtain a mature foot-and-mouth disease virus suspension culture medium; after centrifugation, the virus suspension culture medium is added with a prepared inactivator and inactivated for 28 hours. During the inactivation process, there is no need to add 50% Na2S2O3 to block it, and it can be directly used for subsequent antigen purification production; (3) Plotting the inactivation kinetics curves of different FMDV strains: Add the prepared inactivator to the venom of O / Mya98, AF / 72, and O / MYA98 / BY strains, add 50% Na2S2O3 to block, and measure the TCID at 0, 1, 2, and 3 h of inactivation. 50 The values ​​are expressed with time as the horizontal axis, and the TCID values ​​of O / Mya98, AF / 72, and O / MYA98 / BY strains at 0, 1, 2, and 3 h of inactivation are plotted. 50The values ​​were used as the vertical coordinates, and the inactivation activity curve was drawn to optimize the inactivation process parameters. It was found that the inactivation time of the AF / 72 strain was 15.99-16.47 h, the inactivation time of the O / MYA98 / BY strain was 22.96-24.38 h, and the inactivation time of the O / Mya98 strain was 18.86-18.94 h.

[0027] The specific optimization process of the foot-and-mouth disease virus inactivation process of the present invention is as follows: 1. Optimization of inactivator formulation and establishment of cyclization method, including the following steps: (1) Dissolve BEA in 100 ml of water for injection and NaOH in 50 ml of purified water according to the ratio of 80 g:205 g, 75 g:205 g, 70 g:205 g, and 65 g:205 g per 10,000 ml. Then add the NaOH solution to the BEA solution and adjust the volume to 250 ml. After filtering, divide each group into 100 ml and 150 ml. Keep four groups of 100 ml at 37 ° C for 1 hour, test the pH value, and number them as Y250101①, Y250102①, Y250103①, Y250104①... Repeat for 3 batches. The results are shown in Table 1 and Figure 1 .

[0028] (2) Preparation of 1% β-naphthol violet indicator solution: Dissolve 1 g of β-naphthol violet in sterile water, dilute to 100 ml, mix well, filter through a filter disk, and store at 4°C.

[0029] (3) Add 150ml of the inactivator to each of the four groups in (1) and add 15μL of 1% β-naphthol violet indicator solution, divide each into 50ml, and test the pH value. After filtering the rest, keep it at 37℃ for 1 hour (observe the color change during this period. Within 5-10 minutes, judge whether the cyclization is successful. If there is a color change, divide each into 50ml and test the pH value). Test the pH value. Add 3% inactivator to 1000ml of venom (AF / 72 strain) for 4 hours, inactivate it in an inverted can for 24 hours, add 0.68% of 50% Na2S2O3 to block, test the pH value, send it to the suckling mouse for safety inspection, and test 146S (ug / ml). Repeat three times. The results are shown in Table 2. Figure 2 、 Figure 3 、 Figure 4 . Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 , NaOH and BEA (unit: g / 10,000 mL) in bottles A, B, C, and D are prepared in the ratios of 80g:205g, 75g:205g, 70g:205g, and 65g:205g.

[0030] (4) The four groups of inactivated antigens in (2) were challenged with temperature (37°C, 4 days) and 146S (ug / ml) was detected. The results were repeated three times. See Table 2. (5) The optimal temperature challenge combination from (4) was applied to large-scale production and repeated three times. The results are shown in Table 3.

[0031] (6) Conclusion: The average pH values ​​of the four inactivator formulations in this experiment (unit: g / 10,000 mL) were 9.63, 8.87, 8.45, and 8.10, respectively, after three replicates. After cyclization with β-naphthol violet as an indicator for 10 minutes, the purple color turned pale orange. The average pH values ​​were similar to those obtained with cyclization with β-naphthol violet as an indicator for 10 minutes or with direct cyclization for 1 hour. All formulations passed safety inspection in suckling mice, indicating that the inactivator can successfully cyclize at 37°C for 10 minutes. The average pH value of the inactivated antigen ranged from 7.80 to 7.95, which is close to the pH value of inactivated antigens produced in large-scale production. The three formulations with the 70g:205g formulation exhibited relatively stable 146S stability during temperature challenge, demonstrating the lowest average degradation rate. Therefore, the 70g:205g formulation was selected for subsequent large-scale production validation. During large-scale production, three batches of the inactivator, each 100,000 mL, were prepared. After adding β-naphthol violet as an indicator and cyclizing for 10 minutes, the purple color turned light orange. The pH value was identical to that observed after cyclizing for 10 minutes with β-naphthol violet or directly for 1 hour. Both batches passed safety inspections in suckling mice, demonstrating that the inactivator can successfully cyclize after maintaining the inactivator at 37°C for 10 minutes. After a temperature challenge (37°C for 4 days), the inactivated antigen remained stable for 146 seconds, with a degradation rate of no more than 30%. Therefore, the optimal ratio was 70g:205g (g / 10,000 mL).

[0032] 2. Optimization of inactivation agent storage temperature and time, including the following steps: (1) Culture of BHK-21 adherent cells: BHK-21 cells were revived and cultured in DMEM medium containing 10% NCS (newborn calf serum) and fetal bovine serum as the growth medium. After the cells grew into a monolayer, the medium was discarded and the cells were digested with 0.25% trypsin and passaged for 2 to 3 times.

[0033] (2) Preparation of inactivated samples: The inactivators stored at different temperatures (4°C, 25°C, 37°C) and for different times (2d, 6d, 10d, 14d, 30d) were used to inactivate the O / MYA98 / BY venom for 3 h, and then 0.68% of 50% Na2S2O3 was added to block the venom and mixed thoroughly for later use.

[0034] (3) 96-well plate step dilution method for titer determination: 100 μL of cell suspension was added to each well of the 96-well plate. The cells grew into a monolayer within 24 hours. The cell growth medium was discarded and 100 μL of 2% NCS DMEM was added as the maintenance medium. The inactivated sample was diluted 10-fold in 96-well culture plates (i.e., 10 -1 , 10 -2 , 10 -3 Each well contains 100 μL of virus suspension. Eight wells are prepared for each dilution, with two replicates for each sample. The culture plates are placed in a 5% CO2 incubator at 37°C. Cytopathic effects are observed for 3-4 days. The results are recorded and the TCID is calculated using the Karber method. 50 The results are shown in Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 , Table 4.

[0035] (4) Conclusion: Table 4 shows that the inactivator has the best inactivation effect at different storage temperatures (4°C, 25°C, and 37°C) and storage times (2 days, 6 days, 10 days, 14 days, and 30 days). Furthermore, the inactivator is most stable within 14 days of storage. Therefore, the optimal storage time and temperature for the inactivator are: at 4°C, the storage period is ≤14 days.

[0036] 3. The toxicity test of 50% Na2S2O3 on BHK-21 cells and the optimization of the dosage of 50% Na2S2O3 after inactivation in large-scale production include the following steps: (1) Toxicity test of 50% Na2S2O3 on BHK-21 cells: 50% Na2S2O3 blocking agent was added to the cells in a 96-well plate at a ratio of 2%, 1.8%, 1.6%, and 0.2% (the control group did not add 50% Na2S2O3 blocking agent). The cells were observed and counted after 24 and 48 hours. This was repeated twice. The results are shown in Table 5.

[0037] (2) Optimization of the dosage of 50% Na2S2O3 after optimizing the inactivator formula (pill test): 3% inactivator was added to 1250ml of AF / 72 strain venom for 28 hours. The venom was then divided into five parts and 0%, 0.2%, 0.4%, 0.6%, and 0.68% (production control) of 50% Na2S2O3 were added respectively. The mixture was then sent for testing of 146S (ug / ml) and safety inspection of suckling mice. This was repeated twice. The results are shown in Table 6.

[0038] (3) Optimization of the dosage of 50% Na2S2O3 after optimizing the inactivator formula (large-scale production): 3% of the inactivator was added to the AF / 72 strain venom for 28 hours without adding 50% Na2S2O3. The 146S (μg / ml) was tested and the suckling mice were inspected. This was repeated three times. The results are shown in Table 6.

[0039] (4) Conclusion: Toxicity test of 50% Na2S2O3 on BHK-21 cells: When the dosage of 50% Na2S2O3 is reduced, the cytotoxicity is lower. When the dosage is between 0%-0.2%, the cell viability is ≥100%. Currently, 0.68% of 50% Na2S2O3 is added for blocking.

[0040] Optimizing the dosage of 50% Na2S2O3 after optimizing the inactivator formulation: When the dosage of 50% Na2S2O3 was reduced, the 146S concentration (μg / ml) of the antigen remained the same as when 0.68% 50% Na2S2O3 was added to block the antigen's 146S concentration (μg / ml), and both suckling mice passed safety inspection. Therefore, after adding the inactivator to the venom for 28 hours of inactivation, the addition of 50% Na2S2O3 to block the antigen is unnecessary. Large-scale production has also confirmed that even after adding the inactivator to the venom for 28 hours of inactivation, the addition of 50% Na2S2O3 to block the antigen is not necessary, and the venom can be used directly in subsequent antigen purification production.

[0041] IV. TCID of different FMDV strains (AF / 72, O / MYA98 / BY, and O / Mya98) 50 Establishing an inactivation kinetic curve involves the following steps: (1) TCID of different strains of foot-and-mouth disease virus (AF / 72 strain, O / MYA98 / BY strain, O / Mya98 strain) 50 : Prepare the adherent cells cultured in step 2 (1) for use, and follow the steps in step 2 (3) to measure the TCID of the O / MYA98 / BY strain venom at 0, 1, 2, and 3 hours of inactivation. 50 Repeat (1) to measure the TCID of AF / 72 strain and O / Mya98 strain venom at 0, 1, 2, and 3 hours of inactivation. 50 The results were shown in Table 7.

[0042] (2) Plotting of the inactivation kinetics curves of different strains of foot-and-mouth disease virus: with hours as the horizontal axis, the TCID of the O / MYA98 / BY strain venom inactivated at 0, 1, 2, and 3 hours in (1) 50 The average value of the value is the vertical axis, and the inactivation efficiency curve is drawn using the table function of EXCEL2019. Repeat (2) to draw the inactivation efficiency curve of the AF / 72 strain and the O / Mya98 strain venom. The results are shown in Figure 10 、 Figure 11 、 Figure 12 .

[0043] (3) After 2 months, retest the inactivation curves of different strains and optimize the inactivation process parameters. The results are shown in Table 8. Figure 13 、 Figure 14 、 Figure 15 .

[0044] (4) Conclusion: The inactivation rate of foot-and-mouth disease AF / 72 strain is 0.914-0.935 log, the theoretical inactivation zero point is 8.51-8.81 h, and the inactivation endpoint is 10 -7 The inactivation time of AF / 72 strain is calculated to be 15.99-16.47h. The inactivation rate of O / MYA98 / BY strain of foot-and-mouth disease is 0.604-0.637log, the theoretical inactivation zero point is 11.97-12.79h, and the inactivation endpoint is 10 -7 The inactivation time of O / MYA98 / BY strain is calculated to be 22.96-24.38h. The inactivation rate of foot-and-mouth disease O / Mya98 strain is 0.807-0.81log, the theoretical inactivation zero point is 10.18-10.30h, and the inactivation endpoint is 10 -7 The inactivation time of O / Mya98 strain is calculated to be 18.86-18.94h. The inactivation time in large-scale production is currently 28h, and the safety is also higher than the 10 required by the Veterinary Pharmacopoeia. -6 Based on the above conclusions, it can be concluded that the virus is completely inactivated during the inactivation process of each batch of venom in large-scale production to avoid biosafety risks in vaccine production.

Claims

1. A large-scale production inactivation process for foot-and-mouth disease virus, comprising the following steps: (1) Preparation of the inactivator and establishment of the cyclization method: dissolve dibromoethylamine hydrobromide (BEA) in injection water, add NaOH solution, adjust the ratio of NaOH to BEA, add β-naphthol violet indicator, detect pH value and color change, and judge whether BEA cyclization is successful. When the color changes from purple to light orange and the pH is between 7.7-7.9, it indicates that the inactivator cyclization is successful; the prepared inactivator is stored at 2-8℃, and the shelf life is ≤14 days; (2) Venom inactivation: Use the BHK-21 cell suspension method to culture foot-and-mouth disease virus to obtain a mature foot-and-mouth disease virus suspension culture medium; after centrifugation, the virus suspension culture medium is added with a prepared inactivator for inactivation. During the inactivation process, there is no need to add 50% Na2S2O3 to block it, and it can be directly used for subsequent antigen purification production; (3) Plotting the inactivation kinetics curves of different FMDV strains: Add the prepared inactivator to the venom of O / Mya98, AF / 72, and O / MYA98 / BY strains, add 50% Na2S2O3 to block, and measure the TCID at 0, 1, 2, and 3 h of inactivation. 50 The values ​​are expressed with time as the horizontal axis, and the TCID values ​​of O / Mya98, AF / 72, and O / MYA98 / BY strains at 0, 1, 2, and 3 h of inactivation are plotted. 50 The values ​​were used as the vertical coordinates, and the inactivation activity curve was drawn to optimize the inactivation process parameters. It was found that the inactivation time of the AF / 72 strain was 15.99-16.47 h, the inactivation time of the O / MYA98 / BY strain was 22.96-24.38 h, and the inactivation time of the O / Mya98 strain was 18.86-18.94 h.

2. The large-scale production inactivation process for foot-and-mouth disease virus according to claim 1, characterized in that: In step (1), NaOH and BEA are prepared in a ratio of 70 g:205 g per 10,000 ml.

3. The large-scale production inactivation process for foot-and-mouth disease virus according to claim 1, characterized in that: In step (1), dibromoethylamine hydrobromide is maintained at 37°C for 10 minutes to ensure successful cyclization and improve biosafety.

4. The large-scale production inactivation process for foot-and-mouth disease virus according to claim 1, characterized in that: In step (1), the mass concentration of the β-naphthol violet indicator solution is 1%.

5. The large-scale production inactivation process for foot-and-mouth disease virus according to claim 1, characterized in that: In step (1), the inactivation agent is stored at 4°C for ≤14 days to ensure the inactivation effect and antigen stability.

6. The large-scale production inactivation process for foot-and-mouth disease virus according to claim 1, characterized in that: In step (2), the inactivation time is 28 hours.

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