Purification method of coxsackie virus 16
Through pretreatment of the virus ultrafiltration concentrate and ion exchange gel chromatography, the problem of inefficient purification of Coxsachusetts 16 was solved by using Capto Q Impres gel media, and efficient virus purification and vaccine production optimization were achieved.
Patent Information
- Application Number
- CN202510746242.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-08-22
AI Technical Summary
The prior art is difficult to efficiently isolate and purify Coxsackie virus type 16, resulting in a complex and inefficient production process of hand, foot and mouth disease vaccines.
The pretreatment method of the virus ultrafiltration concentrate was adopted, including the addition of chemical reagents such as Triton-X100, trimethyl citrate and histidine, and incubated at a specific temperature, followed by ion exchange gel chromatography, and purification using Capto Q Impres gel medium.
The recovery rate of purified liquid and impurity protein removal rate of Coxsackie virus type 16 has been improved, the vaccine production process has been simplified, and the pollution risk and cost have been reduced.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of virus vaccines, and in particular to a method for purifying Coxsackievirus type 16. Background Art
[0002] Hand, foot, and mouth disease (HFMD) is a highly contagious viral disease that primarily infects children from spring to autumn. Epidemiologically, the incidence of HFMD in my country has been increasing, with a significant increase in severe cases and deaths. Humans are the sole natural reservoir for enteroviruses, which are highly resilient and resistant to detergents, weak acids, and ether. They are insensitive to 70% ethanol solutions but sensitive to ultraviolet light, chlorine-containing disinfectants, formaldehyde, and strong alkalis. The main pathogens causing the infection are enterovirus 71 (EV71), coxsackievirus 16 (CVA16), and other enteroviruses. In recent years, with the widespread adoption of inactivated EV71 vaccination, the incidence of HFMD caused by EV71 has significantly decreased. Vaccination is an effective preventive measure for HFMD. However, the prevalence of CVA16 remains on the rise, with the number of infections increasing year-on-year. The lack of effective medications and treatments has led to an increase in the proportion of severe cases. The development of a CVA16 vaccine is crucial for the prevention and control of HFMD.
[0003] CVA16 virus particles are approximately 27-30nm in diameter, lack a cell membrane, and exhibit an icosahedral structure. The viral gene encodes four polypeptides (VP1 / 2 / 3 / 4) that form a protomer, which is assembled into subunits with a pentamer-like structure. The 60 subunits interconnect to form the viral capsid. Although the virus particle morphology is very similar to that of EV71, the CVA16 virus is expressed at very low levels in cell culture, making its isolation and purification challenging.
[0004] The general production process of hand, foot and mouth disease vaccine, from harvesting virus culture fluid to preparing stock solution, often has many steps and a complicated process. The stock solution can only be prepared through processes such as centrifugal clarification, ultrafiltration dialysis, PEG precipitation, density gradient centrifugation, molecular sieve chromatography, secondary ultrafiltration, and filtration sterilization.
[0005] Therefore, seeking a simple and efficient method for purifying Coxsackievirus type 16 and optimizing the downstream production process of hand, foot and mouth disease vaccine are important links in promoting the prevention and control of hand, foot and mouth disease. Summary of the Invention
[0006] In order to solve the above technical problems, the present application provides a method for purifying Coxsackievirus type 16.
[0007] In a first aspect, the present application provides a method for purifying Coxsackievirus type 16, comprising sequentially performing the following steps: The virus ultrafiltration concentrate is pre-treated and then subjected to ion exchange gel chromatography to obtain a virus purified solution; The specific steps of the pretreatment are: adding Triton-X100 with a final concentration of 150-250 μg / ml, trimethyl citrate with a final concentration of 30-50 μg / ml, and histidine with a final concentration of 10-20 μg / ml to the virus ultrafiltration concentrate, incubating at 10-18°C for 2-4 hours; then adding glycerol with a final concentration of 10-20 μg / ml, and incubating at 20-30°C for 20-60 minutes; The ion exchange gel chromatography adopts a flow-through process and utilizes Capto Q Impres gel medium for ion exchange gel chromatography.
[0008] The purification method provided in the present application is achieved by using Capto Q Impres filler. Before the purification operation, the virus ultrafiltration concentrate is pretreated. This method can improve the separation and purification effect of CVA16 virus fermentation broth without affecting biological activity, remove impurity proteins, and increase antigen recovery rate.
[0009] Triton X-100 is a nonionic surfactant that increases protein solubility. Its unique molecular structure promotes interactions between target molecules and the solvent, effectively reducing interfacial tension and better controlling the contact area between the liquid and solid phases, thereby improving separation purity. Trimethyl citrate, as a weak organic acid buffer, helps create favorable environmental conditions for the smooth progress of subsequent steps. It also controls charge distribution, allowing negatively charged nucleases to more easily adsorb to the surface of the normal phase medium and be removed. Histidine not only acts as a buffer, stabilizing the pH of the solution, but also binds to impurity proteins, altering their surface charge and solubility, thereby facilitating their separation from the ultrafiltrate. Adding a specific concentration of Triton-X100, trimethyl citrate, and histidine to the viral ultrafiltrate concentrate and incubating at a specific temperature range of 10-18°C for 2-4 hours helps break down interactions between viral particles and impurity proteins, making antigens more easily isolated. This ensures the full effectiveness of the aforementioned chemical reagents while avoiding unnecessary damage to the viral antigens. Afterwards, a specific concentration of glycerol is added for a second incubation. Glycerol has good stability and can protect the antigen from damage during subsequent processing. At the same time, glycerol helps to increase the solubility of the viral antigen, thereby helping to improve the recovery rate of the viral antigen in the subsequent separation and purification steps.
[0010] In the ion exchange gel chromatography step: Capto Q Impres is an ion exchange filler in the Capto filler series. Its basic skeleton is a chemically modified agarose matrix with strong rigidity and fast mass transfer. This series of gels has good mechanical properties, including dynamic binding capacity, pressure flow rate, selectivity, stability, etc., which determine the scalability of the production process. Capto QImpres is a high-load anion exchange filler with a functional group of -CH2-N + -(CH3)3, with a particle size of 40um, has a higher resolution compared to traditional ion exchange fillers.
[0011] Preferably, the method for obtaining the virus ultrafiltration concentrate is: culturing the Coxsackievirus type 16 virus harvest liquid on Vero cells, filtering and clarifying it using a 0.8+0.45μm filter element; and then ultrafiltration and concentrating it 20-50 times through a 300KD membrane package to obtain the virus ultrafiltration concentrate.
[0012] Preferably, the specific steps of the pretreatment are: adding a non-ionic surfactant Triton-X100 with a final concentration of 170-220 μg / ml, trimethyl citrate with a final concentration of 35-45 μg / ml, and histidine with a final concentration of 12-18 μg / ml to the virus ultrafiltration concentrate, and incubating at 12-16 ° C for 2.5-3.5 hours; then adding glycerol with a final concentration of 12-18 μg / ml, and incubating at 23-27 ° C for 30-50 minutes.
[0013] In a specific embodiment, the specific steps of the pretreatment are: adding a non-ionic surfactant Triton-X100 with a final concentration of 200 μg / ml, trimethyl citrate with a final concentration of 40 μg / ml, and histidine with a final concentration of 15 μg / ml to the virus ultrafiltration concentrate, and incubating at 14°C for 3 hours; then adding glycerol with a final concentration of 15 μg / ml and incubating at 25°C for 40 minutes.
[0014] Through experimental analysis, it can be seen that the present application selects the raw material components of the above concentrations and controls the incubation conditions to the above parameter conditions, which can further improve the recovery rate of the target product of the Coxsackievirus type 16 purified liquid and the removal rate of impurity proteins.
[0015] Preferably, the ion exchange gel chromatography comprises the following steps in sequence: column packing with Capto Q Impres gel medium, online disinfection, column equilibration, system setup, sample loading, sample flow-through peak collection, column cleaning and regeneration.
[0016] Preferably, the specific steps of the column packing are: taking Capto Q Impres gel medium, replacing the gel protective liquid with ultrapure water, stirring evenly, adding it to a 16mm×20cm chromatography column, fixing the column bed height to 8-12cm, the column volume to 18-22ml, and balancing with ultrapure water; using 1% acetone to measure the column efficiency to ensure that the column efficiency is greater than 3500 and the symmetry is between 0.8-1.2.
[0017] Preferably, the specific steps of online disinfection are: using NaOH with a concentration of 0.45-0.65 mol / L to disinfect the chromatography column online, with a flow rate of 50-70 cm / h and a disinfection volume of 2-5CV.
[0018] Preferably, the specific step of balancing the chromatography column is: balancing the chromatography column with a phosphate-sodium chloride buffer containing a sodium chloride concentration of 0.15-0.25 mol / L and a pH of 7.2-7.6, with an equilibrium volume of 5-10CV and a flow rate of 50-70 cm / h until the ultraviolet absorption baseline is stable.
[0019] Preferably, the sample loading parameters are: sample loading volume of 50%-150%, and sample loading flow rate of 50-70 cm / h.
[0020] In a specific embodiment, the loading parameter conditions are: the loading volume can be 50%, 75%, 100%, 125%, 150%, and the loading flow rate can be 50cm / h, 55cm / h, 60cm / h, 65cm / h, 70cm / h.
[0021] Preferably, the specific step of collecting the sample flow-through peak is: starting to collect the peak range from when the ultraviolet absorbance value at 280 nm rises, to obtain the first peak flow-through component, which is the Coxsackievirus type 16 purified solution.
[0022] In a second aspect, the present application provides a Coxsackievirus type 16 antigen obtained using the above-mentioned purification method.
[0023] In summary, the technical solution of this application has the following effects: The present application provides a novel CVA16 virus purification method to improve the separation efficiency of the target product and ensure the recovery rate of the target product of the CVA16 virus purification solution and the removal rate of impurities. This application optimizes the downstream production process of hand, foot and mouth disease vaccine, reduces the operating steps in the vaccine preparation process, reduces the risk of contamination, improves production efficiency, and can effectively save costs. DETAILED DESCRIPTION
[0024] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in this application. Example
[0025] Example 1 Example 1 provides a method for purifying Coxsackievirus type 16.
[0026] The purification method of Coxsackievirus 16 in this embodiment specifically comprises the following steps: (1) Obtaining virus ultrafiltration concentrate The Coxsackievirus type 16 virus harvest was obtained by culturing on Vero cells and, under the protection of a clean bench, aseptically connected to the liquid inlet of a clarification filter with a pore size of 0.8+0.45 μm to remove cell debris, larger aggregates, and insoluble impurities; the liquid outlet of the clarification filter was connected to a sterile container, and the peristaltic pump was turned on to collect the viral clarification liquid.
[0027] Using a membrane with a molecular weight cutoff of 300 kD, ultrafiltration and concentration were performed 30-fold. Diafiltration was performed using a phosphate-sodium chloride buffer system with a sodium chloride concentration of 0.15 mol / L and a pH of 7.4. The diafiltration volume was 10 times the concentrate volume, and the ultrafiltration concentrate was collected in a sterile container. The resulting viral ultrafiltration concentrate had an antigen content of 1473.69 U / ml and a protein content of 978.38 μg / ml.
[0028] (2) Pretreatment of the virus ultrafiltration concentrate: Add the non-ionic surfactant Triton-X100 with a final concentration of 200 μg / ml, trimethyl citrate with a final concentration of 40 μg / ml, and histidine with a final concentration of 15 μg / ml to the virus ultrafiltration concentrate, and incubate at 14°C for 3 h; then add glycerol with a final concentration of 15 μg / ml and incubate at 25°C for 40 min.
[0029] (3) CVA16 virus chromatography purification method This method is implemented using Capto Q Impres medium and the specific steps are as follows: (3.1) Column Packing: Capto Q Impres gel medium and XK16 / 20 chromatography columns were purchased from Cytiva. The gel protective solution was replaced with ultrapure water, stirred thoroughly, and then added to a 16 mm × 20 cm chromatography column with a bed height of 10 cm and a column volume (CV) of 20 ml. The column was equilibrated with ultrapure water. The column efficiency was then measured using 1% acetone to ensure a column efficiency greater than 3500 and a symmetry between 0.8 and 1.2.
[0030] (3.2) Online disinfection: Use 0.5 mol / L NaOH to disinfect the chromatography column online at a flow rate of 60 cm / h and a disinfection volume of 2 CV.
[0031] (3.3) Column equilibration: Use phosphate-sodium chloride buffer with a sodium chloride concentration of 0.2 mol / L and a pH of 7.4 to equilibrate the column. The equilibration volume is 7 CV and the flow rate is 60 cm / h until the UV absorption baseline is stable.
[0032] (3.4) System setup: Preheat the chromatography system, set the alarm pressure, column position, injection and collection flow paths, set the UV absorbance to 280 nm, and set aside.
[0033] (3.5) Sample loading: Sterile sample loading, sample volume is 100% column volume, sample loading flow rate is 60 cm / h.
[0034] (3.6) Sample Collection: Collect the flow-through peak, starting when the UV absorbance begins to rise, passing the first absorbance peak, and stopping collection when the UV absorbance begins to rise at the second absorbance peak. This will yield the first peak of the flow-through fraction and send it for testing of antigen and protein content. Simultaneously, collect the second peak of the flow-through fraction using the same method and send it for testing of antigen and protein content.
[0035] (3.7) Column Cleaning and Regeneration: Clean and disinfect the column with 0.5 mol / L NaOH at a flow rate of 60 cm / h and a disinfection volume of 2 column volumes. If the column is not in use for an extended period, store it in 0.1 mol / L NaOH.
[0036] (3.8) Antigen content and protein content results: Antigen recovery rate = (purified solution volume × purified solution antigen content) / (sample volume × virus ultrafiltration concentrate antigen content) × 100%; The impurity protein removal rate was calculated as follows: impurity protein removal rate = 1-(purified solution volume × purified solution protein content) / (sample volume × virus ultrafiltration concentrate protein content) × 100%.
[0037] In this example, the antigen recovery rate of the first peak of the flow-through component was 86.54%, and the protein removal rate was 94.69%. The antigen recovery rate of the second peak of the flow-through component was 10.33%, and the protein removal rate was 69.65%. As can be seen from the results, the antigen recovery rate and protein removal rate of the first peak of the flow-through component were high, but the protein content of the second peak was relatively high, failing to achieve an effective separation effect. Therefore, collecting the first flow-through peak as the purified liquid can achieve the desired separation effect. The peak collection range should start when the UV absorbance value rises, and stop collecting when it rises again after the peak peak drops.
[0038] Example 2-11 Examples 2-11 each provide a method for purifying Coxsackievirus type 16.
[0039] The difference between the above embodiment and embodiment 1 is that the pretreatment method of the virus ultrafiltration concentrate is different, as shown in Table 1.
[0040] Table 1 Pre-treatment parameters of virus ultrafiltration concentrate in Examples 1-11 The remaining parameters of the above embodiment are the same as those of embodiment 1.
[0041] Examples 12-13 Examples 12-13 each provide a method for purifying Coxsackievirus type 16.
[0042] The difference between the above embodiment and embodiment 1 is that the sample loading parameter conditions are different, as shown below.
[0043] In Example 12: the sample loading amount was 100% column volume, and the loading flow rate was 50 cm / h.
[0044] In Example 13: the sample loading amount was 100% column volume, and the loading flow rate was 70 cm / h.
[0045] The other process parameters in the above embodiment are the same as those in Example 1.
[0046] Comparative Example Comparative Example 1 Comparative Example 1 provides a method for purifying Coxsackievirus type 16.
[0047] The difference between this comparative example and Example 1 is that the virus ultrafiltration concentrate was not pre-treated.
[0048] The other process parameters in this comparative example are the same as those in Example 1.
[0049] Comparative Examples 2-3 Comparative Examples 2-3 respectively provide a method for purifying Coxsackievirus type 16.
[0050] The difference between the comparative example and Example 1 is that the pretreatment method of the virus ultrafiltration concentrate is different, as shown below.
[0051] In Comparative Example 2: Pretreatment of the virus ultrafiltration concentrate: To the virus ultrafiltration concentrate were added a non-ionic surfactant Triton-X100 with a final concentration of 200 μg / ml, trimethyl citrate with a final concentration of 40 μg / ml, histidine with a final concentration of 15 μg / ml, and glycerol with a final concentration of 15 μg / ml, and incubated at 25 ° C for 40 min.
[0052] In Comparative Example 3: histidine was replaced by an equal amount of glycine.
[0053] The other process parameters in the above comparative example are the same as those in Example 1.
[0054] Comparative Examples 4-13 Comparative Examples 4-13 respectively provide a method for purifying Coxsackievirus type 16.
[0055] The difference between the comparative example and Example 1 is that the pretreatment method of the virus ultrafiltration concentrate is different, as shown in Table 2.
[0056] Table 2 Pre-treatment parameter conditions of virus ultrafiltration concentrate in comparative examples 4-13 The other process parameters in the above comparative example are the same as those in Example 1.
[0057] Performance testing According to the purification methods of the examples and comparative examples, the first peak of the flow-through fraction and the second peak of the flow-through fraction were collected and sent for detection of antigen content and protein content.
[0058] Test results: as shown in Table 3.
[0059] Table 3 Purification results of Coxsackievirus 16 in Examples 1-13 and Comparative Examples 1-13 Combined with Table 3, by comparing the test results of the embodiment and the comparative example, it can be seen that the virus ultrafiltration concentrate is pre-treated using the technical solution of the present application, and the flow-through process is adopted. The Capto Q Impres gel medium is used for ion exchange gel chromatography, and the first peak of the flow-through component is used as the target product. The separation efficiency of the target product of the Coxsackievirus type 16 purified solution can be effectively improved, and the recovery rate of the target product and the removal rate of the impurity protein can be ensured.
[0060] In Comparative Example 1, the virus ultrafiltration concentrate was not pretreated. In Comparative Example 2, Triton-X100, trimethyl citrate, histidine, and glycerol were added simultaneously when the virus ultrafiltration concentrate was pretreated. In Comparative Example 3, an equal amount of glycine was used instead of histidine when the virus ultrafiltration concentrate was pretreated. In Comparative Example 4, trimethyl citrate and histidine were not added when the virus ultrafiltration concentrate was pretreated. The separation rate of the target product of the Coxsackievirus type 16 purified solution was poor, and the recovery rate of the antigen target product and the removal rate of the impurity protein were low.
[0061] By comparing the test results of Examples 1-5 with Comparative Examples 5-6, it can be seen that the concentrations of Triton-X100, trimethyl citrate, and histidine in Comparative Examples 5-6 are not matched, resulting in a poor separation rate of the target product in the purified solution of Coxsackievirus type 16. In contrast, the Examples of the present application effectively improve the recovery rate of the target product and the removal rate of impurity proteins by precisely matching the concentrations of Triton-X100, trimethyl citrate, and histidine.
[0062] By comparing the test results of Examples 1, 6-7 and Comparative Examples 7-9, it can be seen that the incubation temperature after adding Triton-X100, trimethyl citrate, and histidine in Comparative Example 7 was low, the incubation temperature after adding Triton-X100, trimethyl citrate, and histidine in Comparative Example 8 was high, and the incubation time after adding Triton-X100, trimethyl citrate, and histidine in Comparative Example 9 was short, resulting in a poor separation rate of the target product in the purified Coxsackievirus type 16 solution. In contrast, the examples of the present application effectively improved the recovery rate of the target product and the removal rate of impurity proteins by controlling the incubation conditions to 10-18°C for 2-4 hours.
[0063] By comparing the test results of Examples 1, 8-11 with Comparative Examples 10-13, it can be seen that the concentration of glycerol added in Comparative Examples 10-11 is relatively low or relatively high, the incubation temperature after adding Triton-X100, trimethyl citrate, and histidine in Comparative Example 12 is relatively low, and the incubation time after adding Triton-X100, trimethyl citrate, and histidine in Comparative Example 13 is relatively long, resulting in a poor separation rate of the target product in the purified solution of Coxsackievirus type 16. In contrast, the examples of the present application effectively improve the recovery rate of the target product and the removal rate of impurity proteins by adding glycerol at a final concentration of 10-20 μg / ml and incubating at 20-30°C for 20-60 minutes.
[0064] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.
Claims
1. A method for purifying Coxsackievirus type 16, characterized in that: The method comprises the following steps: pre-treating the virus ultrafiltration concentrate and then performing ion exchange gel chromatography to obtain a virus purified solution; The specific steps of the pretreatment are: adding Triton-X100 with a final concentration of 150-250 μg / ml, trimethyl citrate with a final concentration of 30-50 μg / ml, and histidine with a final concentration of 10-20 μg / ml to the virus ultrafiltration concentrate, incubating at 10-18°C for 2-4 hours; then adding glycerol with a final concentration of 10-20 μg / ml, and incubating at 20-30°C for 20-60 minutes; The ion exchange gel chromatography adopts a flow-through process and utilizes Capto Q Impres gel medium for ion exchange gel chromatography.
2. The method for purifying Coxsackievirus 16 according to claim 1, wherein: The method for obtaining the virus ultrafiltration concentrate comprises: culturing Coxsackie virus type 16 virus harvest liquid on Vero cells, filtering and clarifying the liquid using a 0.8+0.45 μm filter element; and then ultrafiltration and concentrating the liquid 20-50 times through a 300 KD membrane package to obtain the virus ultrafiltration concentrate.
3. The method for purifying Coxsackievirus 16 according to claim 1, wherein: The specific steps of the pretreatment are: adding a non-ionic surfactant Triton-X100 with a final concentration of 170-220 μg / ml, trimethyl citrate with a final concentration of 35-45 μg / ml, and histidine with a final concentration of 12-18 μg / ml to the virus ultrafiltration concentrate, and incubating at 12-16°C for 2.5-3.5 hours; then adding glycerol with a final concentration of 12-18 μg / ml, and incubating at 23-27°C for 30-50 minutes.
4. The method for purifying Coxsackievirus 16 according to claim 1, wherein: The ion exchange gel chromatography comprises the following steps: column packing with Capto Q Impres gel medium, online disinfection, column balancing, system setting, sample loading, sample flow-through peak collection, column cleaning and regeneration.
5. The method for purifying Coxsackievirus 16 according to claim 4, characterized in that: The specific steps of the column packing are: taking Capto Q Impres gel medium, replacing the gel protective liquid with ultrapure water, stirring evenly, adding it to a 16mm×20cm chromatography column, fixing the column bed height to 8-12cm, the column volume to 18-22ml, and balancing with ultrapure water; using 1% acetone to measure the column efficiency to ensure that the column efficiency is greater than 3500 and the symmetry is between 0.8 and 1.
2.
6. The method for purifying Coxsackievirus type 16 according to claim 4, characterized in that: The specific steps of online disinfection are: using NaOH with a concentration of 0.45-0.65 mol / L to disinfect the chromatography column online, with a flow rate of 50-70 cm / h and a disinfection volume of 2-5CV.
7. The method for purifying Coxsackievirus type 16 according to claim 4, characterized in that: The specific steps of balancing the chromatography column are: using a phosphate-sodium chloride buffer containing a sodium chloride concentration of 0.15-0.25 mol / L and a pH of 7.2-7.6 to balance the chromatography column, with an equilibrium volume of 5-10CV and a flow rate of 50-70 cm / h until the ultraviolet absorption baseline is stable.
8. The method for purifying Coxsackievirus type 16 according to claim 4, characterized in that: The sample loading parameters are as follows: sample loading volume is 50%-150%, and sample loading flow rate is 50-70 cm / h.
9. The method for purifying Coxsackievirus type 16 according to claim 4, characterized in that: The specific steps of collecting the sample flow-through peak are: starting to collect the peak range from when the ultraviolet absorption value of 280nm rises, and obtaining the first peak flow-through component, which is the Coxsackie virus type 16 purified liquid.
10. A Coxsackievirus type 16 antigen, characterized in that Obtained by the purification method according to any one of claims 1 to 9.