High-yield PEDV (porcine epidemic diarrhea virus) method
By using a washing liquid containing additives and buffer systems for deep filtration and circulation washing, the problem of precipitated viruses in PEDV vaccines is solved, the harvest rate and purity of the virus are improved, the process is simplified, and the quality of the vaccine is improved.
Patent Information
- Application Number
- CN202411933514.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-02-14
AI Technical Summary
In the preparation process of the pig epidemic diarrhea virus (PEDV) vaccine, some precipitated viruses are unavailable, and the existing process has the disadvantages of long process time, difficult to amplify, many impurities, and difficult process, which limits the quality of the PEDV vaccine. promote.
Using a washing solution including additives and buffer systems, the cell culture containing PEDV was processed by deep filtration, and the cells and precipitates on the filter plate were washed circulatingly, and the washing products were collected and combined with the filtrate to Improve the efficiency of virus harvesting.
It effectively promotes the release and soluble of viruses on the surface of cell membranes and cell debris, improves the harvest rate and purity of viruses, reduces the generation of impurities, simplifies the subsequent purification process, and improves the quality of vaccines.
Smart Images
Figure CN119432777A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological products, and in particular to a method for high-yield PEDV. Background Art
[0002] Currently, porcine epidemic diarrhea virus (PEDV) antigens are cultured and propagated in ST or VERO cells, and the antigen virus titer is harvested to 6-8, but 60% is in the precipitated cells, mainly in the form of aggregates on the cell membrane surface or inside the cell. The precipitated part of the virus is unusable during the vaccine preparation process.
[0003] For insoluble precipitates, the following two methods can be used: 1. Cell disruption method, to release the virus inside the cell and on the membrane surface. After cell disruption, the host protein, host nucleic acid, and host protease inside the cell will be released, resulting in a significant increase in the amount of impurities and increasing the difficulty of the subsequent purification process; 2. Chemical reagent method: using high-concentration guanidine hydrochloride, urea and other chemical reagents to increase solubility, but this method may cause the target to lose activity or bring hidden dangers to the safety of subsequent animal immunization. The above two methods have the disadvantages of long process time, difficulty in scale-up, many impurities, and high process difficulty, which limit the quality improvement of PEDV vaccines. Summary of the invention
[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for high-yield PEDV in view of the shortcomings of the prior art.
[0005] In order to solve the above technical problems, the present invention discloses a method for high-yield PEDV. The specific technical solution is as follows:
[0006] A washing liquid comprising an additive and a buffer system;
[0007] Wherein, the additive is any one or more combinations of sodium chloride, sodium sulfate, Tween 20, arginine, trypsin, urea, transmembrane serine protease, SDS, EDTA or protease inhibitors;
[0008] The buffer system is any one of PBS buffer, HEPES buffer, MEM cell culture medium or DMEM cell culture medium.
[0009] Preferably, the additive is pancreatin or sodium sulfate, and the buffer system is PBS buffer. Further preferably, the additive is pancreatin, and the PBS buffer has a formula of: 20 mM PB, 100 mM NaCl, pH 7.5.
[0010] Wherein, in the washing liquid, the concentration of the additive is 5-200 μg / mL; the pH of the buffer system is 6.0-8.0. Preferably, the concentration of the additive is 5-50 μg / mL, pH 6.5-7.5. More preferably, the concentration of the additive is 20 μg / mL, pH 7.5.
[0011] In a second aspect, the present invention provides a method for high-yield PEDV in the washing solution of the first aspect, the method comprising the step of culturing cells to produce PEDV, the method is one of centrifugation or deep filter plate filtration, preferably deep filter plate filtration, and specifically comprises the following steps:
[0012] (1) deep filtering a cell culture containing PEDV through a filter plate to obtain a filtrate and a filter plate enriched with cells;
[0013] (2) washing the cell-enriched filter plate with the washing solution described in the first aspect, and collecting the washing product;
[0014] (3) The filtrate obtained in step (1) and the washed product obtained in step (2) are combined to obtain a product containing PEDV.
[0015] Wherein, in step (1), the cells are any one of ST cells or Vero cells, preferably Vero cells.
[0016] Wherein, in step (1), the deep filtration has a pressure difference of 0.4-0.6 bar, and the filter plate has a pore size of 5-40 μm. Preferably, the filter plate has a pore size of 12-8 μm.
[0017] Wherein, a filter aid is used for deep filtration, and the added mass of the filter aid is 0.5-1% g / mL of the volume of the cell culture containing PEDV in step (1); the filter aid is preferably diatomaceous earth, and more preferably diatomaceous earth C300 and C100, and the added mass of the filter aid is 1% g / mL of the volume of the cell culture containing PEDV in step (1). More preferably, the filter aid is directly mixed with the cell culture.
[0018] Wherein, in step (2), the washing temperature is 4-35°C and the washing time is 0.5-3h. Preferably, the washing temperature is 4-25°C for 1-2h, and more preferably, the washing temperature is 20°C for 1h.
[0019] Wherein, in step (2), the washing is a filter plate cyclic washing, and the flow rate of the washing liquid is 10-15 mL / min. The filter plate cyclic washing means repeatedly washing the filter plate with the washing liquid.
[0020] Beneficial effects:
[0021] The present invention develops a washing liquid that can promote the release and solubility of the cell membrane surface, the cell fragment surface and the aggregated virus. A deep filtration method is used to treat a cell culture containing PEDV and collect cells and precipitates, separate the filtrate, and ensure that the cells are intact, not only the supernatant free virus is harvested, but also the cells and precipitates are collected on the filter plate. The washing liquid is used to circulate and wash the trapped cells and precipitates on the filter plate to obtain a washing product. The filtrate and the washing product are combined and concentrated, or directly used for inactivation to prepare a finished vaccine.
[0022] Advantages of this technical solution:
[0023] (1) The washing buffer is mild and has no effect on the biological activity of the antigen, but the toxicity can be detected.
[0024] (2) The deep filter plate retains the cell nuclear precipitate without destroying the cells. The host proteins, host nucleic acids, and host proteases in the cells are not released, and less impurities are produced, which makes it easier to improve the purity of the antigen.
[0025] (3) Compared with the existing process, the recovery rate of the target product can be increased by 1.4-4.6 times.
[0026] (4) The turbidity of the product can be effectively controlled, facilitating the subsequent purification process. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.
[0028] Figure 1 The following are microscopic images of cells before and after washing with washing buffer. Figure 1 A is the microscopic inspection picture before washing. Figure 1 B is the microscopic examination after washing.
[0029] Figure 2 This is a data graph showing the effect of additive components in the washing solution on the increase of N protein and TCID50 / mL.
[0030] Figure 3 A line graph showing the mean values of each factor level. DETAILED DESCRIPTION
[0031] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.
[0032] In the following examples, the PEDV virus strains, unless otherwise specified, were isolated from the laboratory of Tiankang Biopharmaceutical Technology Center.
[0033] In the following examples, the PBS buffer, unless otherwise specified, has the following composition: 20 mM PB, 100 mM NaCl, pH 7.5.
[0034] In the following examples, the TCID 50 The detection method is: using TCID 50 To determine the virus content, the sample to be tested was diluted 10 times in MEM culture medium. -1 , 10 -2........... 10 -8 100 μL of each dilution was added to a 96-well cell culture plate, followed by the addition of a trypsin-EDTA-digested Vero cell suspension (i.e., adherent Vero cells were digested with 0.25% trypsin-0.02% EDTA in the culture volume, and the cells were converted from adherent to suspended cells). 100 μL per well (cell content was 3×10 5 Each dilution was repeated 9 times, and a normal cell control was set up. The cells were placed in a 5% CO2 incubator at 37°C. The cytopathic effect and the control were observed daily for 2-5 days. The number of wells with cytopathic effect was recorded, and the TCID of the virus was calculated according to the Reed-Muench method. 50 When calculating the yield and increment, it is important to convert the lgTCID50 / mL value into scientific notation before performing the calculation.
[0035] The N protein detection method is as follows: the N protein in the PEDV antigen sample is detected by the colloidal gold method, the PEDV standard is taken, and the lysate in the kit is used for multiple dilution, and the standard curve is fitted with the corresponding gray value measured by the N protein standard concentration and the detection card. The sample to be tested is diluted to an appropriate concentration with the lysate, the gray value is measured with the colloidal gold reagent card, the gray value is brought into the standard curve, and the N protein content in the sample to be tested is calculated.
[0036] The total protein detection method is as follows: the total protein concentration in the sample is determined by the BCA method, a bovine serum albumin (BSA) standard is taken, and diluted to a series of gradients with a buffer solution, and the sample to be tested is diluted to an appropriate concentration with the same buffer solution. 25 μL of the BSA standard dilution solution and the sample to be tested are added to a 96-well plate, 100 μL of the working solution in the kit is added, and the plate is incubated at 37°C for 30 minutes. The OD value is detected by an enzyme reader at 562 nm, and the standard curve is fitted with the concentration of the standard and the corresponding OD, and the OD value of the sample to be tested is brought into the standard curve to calculate the protein content in the sample to be tested.
[0037] The cell disruption rate is calculated as follows: cell disruption rate = raw material cell density - treated cell density / raw material cell density. The number of cells is determined by trypan blue staining counting method. The sample to be tested is mixed with 0.04% (w / v) trypan blue staining solution at a ratio of 1:1 v / v. The cells are counted under a microscope. Dead cells will be stained blue, and live cells will be transparent and colorless. The cell disruption rate is calculated based on the change in the total number of cells.
[0038] Example 1
[0039] The method for extracting PEDV described in this embodiment (test tube centrifugation method) specifically includes the following steps:
[0040] (1) Separation of free PEDV virus from cells: ST or Vero are suspension cells. After the cells are revived, they are propagated to the third generation in a shake flask. The cells are in good condition and are propagated in a 2L reactor. After growing for 48 hours, PEDV seed virus is taken and inoculated at an MOI of 0.1. After culturing for 24 to 36 hours, the PEDV antigen mixture (i.e., cell culture containing PEDV) is harvested and placed in a sterile centrifuge tube. Centrifuge at 3000g for 10 minutes to separate the cells from the free PEDV virus in the supernatant. The supernatant is retained and the N protein and virus content in the mixture and the centrifuged supernatant (original supernatant) are measured.
[0041] (2) Washing cells with washing solution: add washing buffer (PBS buffer) with the same volume as the antigen mixture to the cell pellet, gently mix at 25°C for 30 minutes, centrifuge at 3000g for 10 minutes, discard the cells, and retain the supernatant as the washing product. The washing product is the supernatant containing PEDV virus. Use an inverted microscope to examine the integrity of the cells before and after washing. Figure 1 The N protein and virus content of the supernatant obtained by PBS washing and centrifugation were measured, as shown in Table 1.
[0042] Table 1 PBS washing precipitate
[0043]
[0044] Example 2 Screening of washing liquid types
[0045] The washing liquid components were screened by test tube centrifugation according to the method described in Example 1. The washing liquid components include a buffer system and one or more combinations of the following additives.
[0046] The concentration of the additive in the washing liquid is any one of the following aj:
[0047] a. 10-300 mM sodium chloride;
[0048] b. 10-300 mM sodium sulfate;
[0049] c. 1%-2% v / v Tween 20;
[0050] d. 50-200 mM arginine;
[0051] e. 10-50 μg / mL pancreatin;
[0052] f. 50-200 mM urea;
[0053] g.10-50 μg / mL transmembrane serine protease (TMPRSS);
[0054] h. 1%-2% m / v (g / mL) SDS;
[0055] i.1-10mM EDTA;
[0056] j. 1-10 mM protease inhibitor (purchased from Bio-Time, catalog number P1006);
[0057] Wherein, the buffer system is PBS buffer.
[0058] The cells were washed with the above-mentioned washing solution at 25°C for 1 h.
[0059] This example screened a total of 10 additives, including sodium chloride, sodium sulfate, Tween 20, arginine, pancreatin, urea, transmembrane serine protease, SDS, EDTA, protease inhibitors, etc. The experimental results are shown in Table 2 and Figure 2 As shown, considering the increase of N protein and toxin titer, it can be determined that pancreatic enzyme can increase the toxin titer and N protein of the supernatant of harvested antigen centrifugation, followed by sodium sulfate. Although the component containing arginine can increase N protein by 158%, it has a serious effect on cells, cell fragmentation (arginine is alkaline, causing cell fragmentation, release of N protein inside the cell, resulting in an increase of 158%), and the toxin titer decreased by 2 titers.
[0060] Table 2 Effect of different additive components in the washing liquid
[0061]
[0062] Example 3 Screening of washing conditions
[0063] In this embodiment, the concentration of the additive in the washing liquid, the washing time, the washing temperature and other conditions are screened and optimized.
[0064] (1) Concentration of additives
[0065] The concentration (5-200 μg / mL) of the key component (additive pancreatin) of the optimal washing solution screened in Example 2 was screened and optimized, and the washing time was 1 h, the washing temperature was room temperature (22° C.), and the other conditions were the same as in Example 1. The test results are shown in Table 3, and the added concentration of pancreatin is preferably 5-50 μg / mL. When the concentration of the pancreatin component in the washing solution is 20 μg / mL, the N protein increment of the washing product is the largest, the toxic value increment can reach 100%, and 1 times the volume of the toxic value of the feed liquid can be recovered. Therefore, the optimal washing solution is used for subsequent screening and optimization experiments, and the components of the optimal washing solution are: PBS buffer and 20 μg / mL pancreatin.
[0066] Table 3 Different concentrations of trypsin washing precipitate
[0067]
[0068] Note: Original supernatant: the supernatant obtained by direct centrifugation of the PEDV antigen mixture; N protein increment = washing product / original supernatant; the increment of the toxin titer is converted into scientific notation for calculation, and the increment of the toxin titer of the washing supernatant with a pancreatin concentration of 5 μg / mL = 10 6.58 / 10 6.80 .
[0069] (2) Washing time
[0070] The washing solution E was composed of PBS buffer + 20 μg / mL pancreatin, and the washing effects of different washing times (0.5, 1, 1.5, 2, 2.5, 3 h) were tested at room temperature. The washing effect was screened according to the increase of N protein in the supernatant. As shown in Table 4, 1-1.5 h was preferred, with an N protein increase of 327-417%, and 1 h was the most preferred.
[0071] Table 4 Protein increments of N at different washing times with washing buffer E
[0072]
[0073] (3) Washing temperature
[0074] The optimal washing solution E was used for washing at 4°C, room temperature (25°C), and 35°C for 1 h. As shown in Table 5, the temperature with the highest washing increment should be 4°C, at which time the N protein increment can reach 157.2%. The N protein increment when washing at room temperature was 140.3%. Considering the high energy consumption and the difficulty in achieving large-scale production, room temperature washing was selected.
[0075] Table 5 N protein increment after washing for 1 h at different temperatures of washing solution
[0076]
[0077] (4) Orthogonal experiment with three factors and three levels: detergent concentration, washing time, and action temperature
[0078] As shown in Table 6, in the three-factor three-level orthogonal experiment of trypsin concentration, washing time and action temperature, the comparison of the factor range values R shows that the degree of influence on the growth of N protein is enzyme action time > temperature > enzyme concentration.
[0079] Depend on Figure 3 From the average line graph of each factor level and Table 6, it can be seen that when the enzyme action temperature is 20℃, the N protein content is the highest; the enzyme action time is optimal when it is 1h, and the enzyme concentration is optimal when it is 20μg / mL. Therefore, the optimal action condition is: 20μg / mL trypsin is acted at 20℃ for 1h.
[0080] Table 6 Orthogonal experiment of three factors and three levels of pancreatin concentration, washing time and action temperature
[0081]
[0082] Example 4
[0083] The extraction method of PEDV described in this embodiment (deep filtration method) specifically includes the following steps:
[0084] (1) Separation of free PEDV virus from cells: The PEDV antigen mixture was harvested according to the method described in step (1) of Example 1, and deep filtration (filter plate pore size 5-20 μm, 2% m / vg / mL diatomaceous earth C300, pressure difference 0.4-0.6 bar) was performed to separate the cells from the free virus in the filtrate. The turbidity of the filtrate was measured during the process, and the presence of cells or cell fragments was examined under a microscope. The pH, conductivity, turbidity, total protein, N protein, and virus content of the cultured antigen and the filtered antigen were measured.
[0085] (2) Washing cells with washing solution: The filter plate intercepts the enriched cells and precipitate (i.e., a filter plate enriched with cells is obtained), and the cells and precipitate are washed by filter plate circulation (i.e., the filter plate is washed repeatedly). The washing temperature is (25-37°C), the washing time is 1h, the flow rate is 10mL / min, the flux is 10LMH, and the pressure difference during the washing process is <0.3bar. The turbidity of the washing filtrate (washing product) is measured during the process, and the presence of cell debris filtered out is examined under a microscope. The pH, conductivity, turbidity, total protein, N protein, and virus content of the washed antigen are measured.
[0086] The filter plate washing process, including the filter plate pore size, filter aid type and dosage, pressure difference, flow rate, etc., was screened and optimized. Turbidity, flux, yield, etc. were investigated.
[0087] First, this embodiment screened 4 kinds of filter plates: Fito AF / CH 6(p) 35-15μm, Fito AF / CH 20(p) 15-6μm, Fito AF / CH 31(p) 12-5μm, Cobet (pore size 5-40μm), considering the back-end turbidity, pressure, and cell penetration, the filter plate pore size 12-8μm was selected, the filter plate alone could not intercept cells, and the filter aid diatomaceous earth was added to the cell-containing harvested liquid, and both diatomaceous earth C300 and C1000 were available. Solid powdered diatomaceous earth was directly added, and the amount added was 0.5-1% (m / V, g / mL) of the PEDV antigen mixture. The amount of diatomaceous earth added was selected according to the adsorption amount of diatomaceous earth on N protein, wherein 0.5-1% adsorption amount was within 10%, preferably 0.5-1.0%. When 0.5% C300 was used for filtration, the results were shown in Table 7, wherein the Cobite filter plate with a filter diameter of 12-8 μm had the best washing effect.
[0088] This embodiment uses 1% C300 plus a 12-8μm filter plate for filtration, and the turbidity of the filtrate is less than 40NTU, and the turbidity of the washing product is less than 20NTU. It meets the requirements for subsequent purification and vaccine preparation. According to the technical scheme optimized in this embodiment, the antigen is harvested, and the cell disruption rate is less than 10%. In addition to the recoverable antigen supernatant (toxin titer 6.5-7.5), the volume of washed antigen (toxin titer 6.5-7.3) can be increased by 1 times, and the N protein yield is increased by 20-50% compared with freeze-thaw crushing; the obtained antigen has less impurities (total protein <2000μg / mL) and turbidity <40NTU, which is lower than the freeze-thaw crushing harvested antigen (turbidity 150-200NTU).
[0089] Table 7 Parameters of filter plate enriched cells
[0090]
[0091] Example 5
[0092] This example is divided into a suspension group, a control group and a test group to verify the yield of different batches of PEDV antigens.
[0093] The preparation method of each group is as follows:
[0094] Suspension group: ST or Vero cells were cultured with a PEDV antigen mixture as described in step (1) of Example 1 to prepare the suspension group.
[0095] Control group: According to the method described in step (1) of Example 1, ST or Vero cells were cultured with a PEDV antigen mixture, the PEDV antigen mixture was not washed, and centrifuged at 10000g for 2 min. The supernatant obtained was the control group, and its N protein and toxin titer were determined. The yield of the control group was the proportion of PEDV antigen harvested from the control group in the PEDV antigen mixture.
[0096] Test group: The PEDV antigen was harvested according to the optimized deep filtration method described in Example 4 as the test group. The test group was divided into the test group filtrate and the test group washing product. The yield of the test group described in this example was the total yield of the filtrate in step (1) and the washing product in step (2) in Example 4.
[0097] This example verifies different batches of PEDV antigens, 3 different strains (PEDV DB strain, PEDV MJ strain and PEDV NT strain, all from Tiankang Pharmaceutical Co., Ltd.), totaling 20 batches. The experimental results are shown in Tables 8 and 9. The increase in washed N protein is >14 μg / mL, and the washed toxicity is >6.15. Compared with the existing process (control group), the yield of N protein is increased by 1.5-4.6 times, and the toxicity yield is increased by 1.4-1.6 times. The turbidity of the control group after centrifugation is 120NTU, and the turbidity after deep filtration is <40NTU. The average total protein of the control group is 2403μg / mL, and the total protein of the experimental group is 1804μg / mL, and the impurity content is 25% lower than that of the control group.
[0098] Table 8 N protein status of different batches of validation recovery products
[0099]
[0100] Table 9 Toxicity of different batches of verified recovered products
[0101]
[0102] Note: The calculation of yield requires converting the LgTCID50 / mL value into scientific notation.
[0103] This example also uses a disruption method to recover antigens in cell precipitates and evaluate N protein and toxicity. The PEDV-DB antigen mixture is obtained as a suspension sample according to the method described in step (1) of Example 1. After centrifugation, a centrifugal supernatant and a precipitate are obtained. The precipitate is resuspended in MEM medium and then subjected to any of the following treatments:
[0104] (1) Ultrasonic disruption: 4°C ice bath, 50 Hz, ultrasonic for 3 seconds, stop for 5 seconds, total ultrasonic time 5 minutes, microscopic examination showed that the cells were completely disrupted;
[0105] (2) Freeze-thaw disruption: Freeze at -20°C for 4 h, thaw in a 37°C water bath, and repeat freeze-thaw three times. Microscopic examination showed that the cells were completely disrupted.
[0106] As shown in Table 10, after the precipitate was crushed by the above two crushing methods, N protein could be recovered, but the toxicity of the recovered product was ≤4.5 (the detection limit was 4), indicating that the recovered product had no toxicity. Related data also showed that PEDV was assembled in cells and mature virus particles were released through exocytosis. The PEDV in cells was not fully assembled and had no immunogenicity. Therefore, the crushing method could not recover more viruses.
[0107] It can be seen that the PEDV inside the cell is not fully assembled, has no immunogenicity, and has no recovery value, so the washing solution of the present invention only recovers the surface of intact cells, or releases the aggregated antigens on the outside.
[0108] Table 10 N protein and toxin titer of cell pellet fragmentation
[0109]
[0110] The washing buffer, deep filter plate enrichment cell and washing method designed in the present invention can effectively improve the yield of the target product, and the impurity protein and turbidity are low, which is conducive to the recovery and preparation of PEDV project viruses. Compared with the existing process (control group), the method of the present invention has the following advantages:
[0111] (1) Double the volume of effective antigen (N protein 14-28μg / mL, toxin titer 6-6.5).
[0112] (2) Turbidity was reduced by 67%.
[0113] (3) Total protein decreased by 25%.
[0114] The present invention provides a method and idea for a high-yield PEDV method. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.
Claims
1. A washing liquid, characterized in that The washing liquid includes additives and a buffer system; Wherein, the additive is any one or more combinations of sodium chloride, sodium sulfate, Tween 20, arginine, trypsin, urea, transmembrane serine protease, SDS, EDTA or protease inhibitors; The buffer system is any one of PBS buffer, HEPES buffer, MEM cell culture medium or DMEM cell culture medium.
2. The cleaning liquid according to claim 1, characterized in that The additive is pancreatin or sodium sulfate; the buffer system is PBS buffer.
3. The cleaning liquid according to claim 1, characterized in that In the washing liquid, the concentration of the additive is 5-200 μg / mL; and the pH of the buffer system is 6.0-8.
0.
4. The cleaning liquid according to claim 3, characterized in that In the washing solution, the concentration of the additive is 5-50 μg / mL.
5. A method for high-yield PEDV, comprising the step of culturing cells to produce PEDV, characterized in that: The PEDV is obtained by washing with the washing liquid according to any one of claims 1 to 4, which specifically comprises the following steps: (1) deep filtering a cell culture containing PEDV through a filter plate to obtain a filtrate and a filter plate enriched with cells; (2) washing the cell-enriched filter plate with the washing solution according to any one of claims 1 to 4, and collecting the washing product; (3) The filtrate obtained in step (1) and the washed product obtained in step (2) are combined to obtain a product containing PEDV.
6. The method according to claim 5, characterized in that In step (1), the cells are ST cells or Vero cells.
7. The method according to claim 5, characterized in that In step (1), the deep filtration has a pressure difference of 0.4-0.6 bar, and the filter plate has a pore size of 5-40 μm.
8. The method according to claim 5, characterized in that In step (1), a filter aid is used for deep filtration, and the added mass of the filter aid is 0.5-1% g / mL of the volume of the cell culture containing PEDV in step (1); the filter aid is preferably diatomaceous earth.
9. The method according to claim 5, characterized in that In step (2), the washing temperature is 4-35°C and the washing time is 0.5-3h.
10. The method according to claim 5, characterized in that In step (2), the washing is a cyclic washing. The flow rate of the washing solution was 10-15 mL / min.