CN115651917A - Purification method of CA16 virus inactivation liquid and preparation method of CA16 virus inactivation stock solution - Google Patents
Purification method of CA16 virus inactivation liquid and preparation method of CA16 virus inactivation stock solution Download PDFInfo
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Abstract
The invention provides a purification method of a CA16 virus inactivation liquid and a preparation method of a CA16 virus inactivation stock solution, belonging to the technical field of biological medicines. The purification method of the CA16 virus inactivation liquid provided by the invention comprises the following steps: and purifying the CA16 virus inactivation solution by adopting a cation exchange chromatography column to obtain a CA16 virus inactivation stock solution. The invention purifies the CA16 virus inactivation liquid based on a cation exchange method, has the same concentration effect on the antigen in the virus inactivation liquid, can remove impurities which cannot be removed by ultrafiltration purification, particularly can obviously reduce the protein content, and improves the purity of effective antigen, thereby improving the quality of a CA16 virus inactivation stock solution product.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a purification method of a CA16 virus inactivation liquid and a preparation method of a CA16 virus inactivation stock solution.
Background
Hand-foot-and-mouth disease (HFMD) is an infectious disease that seriously affects the health of infants, coxsackie virus group 16 (coxsackievirus a16, CA 16), one of the members of the enterovirus family, is another important pathogen causing HFMD besides enterovirus 71 (enterovirus 71, ev71), and has been causing the prevalence or outbreak of HFMD in a plurality of countries or regions along with EV71 virus. Since 2016, with the marketing of inactivated EV71 vaccine, CA16 virus was likely to be the main pathogen causing hand-foot-and-mouth disease in children.
However, the pathology of CA16 virus infection and the related immunological mechanism are not clear, so that the vaccine research is difficult. The preparation of the inactivated CA16 virus stock solution is an important link in the development process of the inactivated virus vaccine, and the purification process of the CA16 virus becomes an important link for enriching effective antigen substances and improving the content of effective antigens.
At present, the preparation process of the CA16 virus inactivation stock solution mainly comprises the steps of CA16 virus proliferation and concentration, molecular sieve chromatography purification, purification solution inactivation and inactivation solution ultrafiltration purification. In the process, ultrafiltration membranes with the molecular weight cutoff of 100KD are adopted in the concentration process after the CA16 virus is proliferated and the ultrafiltration purification process of the inactivated solution, and because the size and the property of the removed impurities are similar, the removal of the impurities in the ultrafiltration purification process of the inactivated solution is limited, and the quality of a CA16 virus inactivation stock solution product is to be improved.
Disclosure of Invention
The invention aims to provide a purification method of a CA16 virus inactivation liquid and a preparation method of a CA16 virus inactivation stock solution, and by adopting the purification method provided by the invention, the purification method has the same concentration effect on antigen in the virus inactivation liquid, and meanwhile, the protein content can be obviously reduced, and the quality of a CA16 virus inactivation stock solution product is improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a purification method of a CA16 virus inactivation solution, which comprises the following steps:
and purifying the CA16 virus inactivation solution by adopting a cation exchange chromatography column to obtain a CA16 virus inactivation stock solution.
Preferably, the exchange packing of the cation exchange chromatography column comprises POROS TM CEX or SP Bestarose High Performance.
Preferably, the purification comprises sequentially performing a first equilibration, a first loading, a second equilibration and a first elution.
Preferably, the first equilibrium solution adopted by the first equilibrium is PBS buffer solution, the concentration of the first equilibrium solution is 0.8-1.5 mmol/L, and the pH value is 7.0-7.4; the volume of the first equilibrium liquid is 3-5 column volumes.
Preferably, the volume of the first loading is 1 to 4 column volumes.
Preferably, the second equilibrium solution adopted by the second equilibrium is PBS buffer solution, the concentration of the second equilibrium solution is 0.8-1.5 mmol/L, and the pH value is 7.0-7.4; the volume of the second equilibrium liquid is 1-3 column volumes.
Preferably, the eluent used for the first elution is sodium chloride aqueous solution or PBS buffer solution containing sodium chloride; the concentration of the sodium chloride aqueous solution is 0.4-1 mol/L; the concentration of the PBS buffer solution in the PBS buffer solution containing sodium chloride is 5-10 mmol/L, and the concentration of the sodium chloride is 0.4-1 mol/L.
Preferably, the first elution further comprises ultraviolet detection, and an eluent corresponding to an absorption peak obtained under the condition that the ultraviolet detection wavelength is 280nm is collected in the first elution process and used as a CA16 virus inactivation stock solution.
The invention provides a preparation method of a CA16 virus inactivation stock solution, which comprises the following steps:
sequentially proliferating and concentrating the CA16 virus to obtain a CA16 virus concentrated solution;
pre-purifying the CA16 virus concentrated solution by using a molecular sieve chromatographic column to obtain a CA16 virus purified solution;
inactivating the CA16 virus purified solution to obtain a CA16 virus inactivated solution;
and purifying the CA16 virus inactivation solution by adopting the purification method in the technical scheme to obtain a CA16 virus inactivation stock solution.
Preferably, the pre-purification comprises sequentially performing a third equilibration, a second loading and a second elution; and the second elution also comprises ultraviolet detection, and in the second elution process, the eluent between two absorption peaks obtained under the condition that the ultraviolet detection wavelength is 280nm is collected and used as the CA16 virus purification solution.
The invention provides a purification method of a CA16 virus inactivation liquid, which comprises the following steps: and purifying the CA16 virus inactivation solution by adopting a cation exchange chromatography column to obtain a CA16 virus inactivation stock solution. The invention purifies the CA16 virus inactivation liquid based on the cation exchange method, has the same concentration effect on the antigen in the virus inactivation liquid, can remove the impurities which cannot be removed by ultrafiltration purification, particularly can obviously reduce the protein content, improves the purity of the effective antigen, is beneficial to improving the specific activity of the CA16 virus inactivation stock solution, and thus improves the quality of the CA16 virus inactivation stock solution product.
Drawings
FIG. 1 is an HPLC chart of the virus-inactivated purified solution prepared in comparative example 1;
FIG. 2 is an HPLC chart of the purified virus inactivation solution prepared in example 1.
Detailed Description
The invention provides a purification method of a CA16 virus inactivation solution, which comprises the following steps:
and purifying the CA16 virus inactivation solution by adopting a cation exchange chromatography column to obtain a CA16 virus inactivation stock solution.
The invention purifies the CA16 virus inactivation liquid by adopting the cation exchange chromatography column, has the same concentration effect on the antigen in the virus inactivation liquid, can remove impurities which cannot be removed by ultrafiltration purification, particularly can obviously reduce the protein content, and improves the purity of effective antigen, thereby improving the quality of a CA16 virus inactivation stock solution product. The source of the CA16 virus inactivation solution is not particularly limited, and the CA16 virus inactivation solution can be prepared by a method well known to those skilled in the art; specifically, the source of the CA16 virus inactivation solution of the present invention will be described in detail later.
In the present invention, the exchange packing of the cation exchange chromatography column preferably comprises POROS TM CEX or SP Bestarose High Performance.
In the present invention, the purification preferably comprises sequentially performing the first equilibration, first loading, second equilibration and first elution, as described in detail below.
In the invention, the first balancing solution used for the first balancing is preferably PBS buffer solution; the concentration of the first equilibrium liquid is preferably 0.8-1.5 mmol/L, and more preferably 1-1.2 mmol/L; the pH value is preferably 7.0-7.4; the volume of the first equilibrium liquid is preferably 3 to 5 column volumes, and more preferably 4 to 5 column volumes. In the present invention, the linear flow rate of the first balancing liquid is preferably 1 to 2cm/min, more preferably 1 to 1.5cm/min. In the present invention, the first balance serves to balance the cationic chromatographic medium, creating a condition of low ionic strength for loading, facilitating binding of the virus particles to the cationic chromatographic medium.
In the present invention, the volume of the first sample is preferably 2 to 4 column volumes, more preferably 3 column volumes; the linear flow rate of the first sample application is preferably 1 to 2cm/min, more preferably 1 to 1.5cm/min.
In the invention, the second balancing solution used in the second balancing is preferably PBS buffer solution; the concentration of the second equilibrium liquid is preferably 0.8-1.5 mmol/L, and more preferably 1mmol/L; the pH value is preferably 7.0-7.4; the volume of the second equilibrium liquid is 1 to 3 column volumes, more preferably 2 to 3 column volumes. In the present invention, the linear flow rate of the second equilibrium liquid is preferably 1 to 2cm/min, more preferably 1 to 1.5cm/min. In the present invention, the second equilibrium is used to wash away impurities that cannot be bound to the cationic chromatographic medium, thereby facilitating the collection of a purer virus sample by subsequent elution.
In the present invention, the eluent used for the first elution is preferably an aqueous sodium chloride solution or a PBS buffer solution containing sodium chloride; the concentration of the sodium chloride aqueous solution is preferably 0.4-1 mol/L, and more preferably 0.5-0.6 mol/L; the concentration of the PBS buffer solution in the PBS buffer solution containing sodium chloride is preferably 5-10 mmol/L, more preferably 5-7 mmol/L, and the concentration of the sodium chloride is preferably 0.4-1 mol/L, more preferably 0.5-0.6 mol/L; the linear flow rate of the eluent in the first elution is preferably 1 to 2cm/min, and more preferably 1 to 1.5cm/min. In the invention, the first elution preferably further comprises ultraviolet detection, and an eluate corresponding to an absorption peak obtained under the condition that the ultraviolet detection wavelength is 280nm is preferably collected as a CA16 virus inactivation stock solution in the first elution process. The invention preferably adopts the eluent of the kind mentioned above, and the subsequent removal is convenient and has no residue.
The invention provides a preparation method of a CA16 virus inactivation stock solution, which comprises the following steps:
sequentially proliferating and concentrating the CA16 virus to obtain a CA16 virus concentrated solution;
pre-purifying the CA16 virus concentrated solution by adopting a molecular sieve chromatographic column to obtain a CA16 virus purified solution;
inactivating the CA16 virus purification solution to obtain a CA16 virus inactivation solution;
and purifying the CA16 virus inactivation solution by adopting the purification method of the technical scheme to obtain a CA16 virus inactivation stock solution.
The CA16 virus is propagated and concentrated in sequence to obtain the CA16 virus concentrated solution. According to the invention, preferably, the CA16 virus seeds are inoculated to KMB17 cells according to the calculated MOI value, and cultured until the KMB17 cells are completely shrunken to obtain CA16 virus liquid; the inoculation is preferably based on 0.1-0.25 MOI, more preferably 0.15-0.2 MOI; the KMB17 cells are preferably KMB17 cells grown in a monolayer; the temperature of the culture is preferably 37 +/-0.5 ℃, and the time is preferably 36-80 h, more preferably 48-72 h, particularly on the basis of complete lesion.
After the CA16 virus liquid is obtained by the proliferation, the CA16 virus liquid is concentrated to obtain CA16 virus concentrated liquid. In the present invention, it is preferable that the concentration further comprises filtering the CA16 virus solution; the filtration preferably comprises deep filtration and bacteria-reduction filtration in sequence; the invention preferably employs a 0.45 μm depth filter for the depth filtration and a 0.45 μm bacteria-reducing grade filter for the bacteria-reducing grade filtration. According to the invention, an ultrafiltration membrane package with the molecular weight cutoff of 100KD is preferably adopted for the concentration, the filter membrane of the ultrafiltration membrane package is preferably washed by PBS buffer solution in the concentration process, and the concentration of the PBS buffer solution is preferably 2-5 mmol/L, and more preferably 2-4 mmol/L; the concentration is preferably 50 to 80 times, more preferably 60 to 70 times.
After the CA16 virus concentrated solution is obtained, the CA16 virus concentrated solution is pre-purified by adopting a molecular sieve chromatographic column to obtain a CA16 virus purified solution. In the present invention, the molecular sieve packing of the molecular sieve chromatography column preferably comprises Sepharose 6FF or Bestarose6FF; the length of the molecular sieve chromatographic column is preferably 80-90 cm, and more preferably 86-90 cm. In the present invention, the pre-purification preferably comprises sequentially performing the third equilibration, the second loading and the second elution.
In the invention, the third balancing solution used for the third balancing is preferably PBS buffer solution; the concentration of the third equilibrium liquid is preferably 2-5 mmol/L, and more preferably 2-3 mmol/L; the pH value is preferably 7.0 to 8.0, more preferably 7.0 to 7.4; the volume of the third equilibrium liquid is preferably 1 to 3 column volumes, and more preferably 2 to 3 column volumes. In the present invention, the linear flow rate of the third equilibration fluid is preferably 0.255 to 0.510cm/min, more preferably 0.255 to 0.355cm/min. In the present invention, the third equilibrium is used to balance the chromatography medium, creating a neutral condition for loading, and facilitating the purification of the virus concentrate.
In the present invention, the volume of the second loading is preferably 3 to 5% of the column volume, more preferably 4 to 5% of the column volume; the linear flow rate of the second sample is preferably 0.255 to 0.510cm/min, more preferably 0.2555 to 0.355cm/min.
In the invention, the eluent used in the second elution is preferably PBS buffer solution, and the concentration of the eluent is preferably 2-5 mmol/L, and more preferably 2-3 mmol/L; the linear flow rate of the eluent in the second elution is preferably 0.255 to 0.510cm/min, and more preferably 0.2555 to 0.355cm/min. In the present invention, the second elution preferably further includes ultraviolet detection, and during the second elution, an eluate between two absorption peaks obtained under the condition that the ultraviolet detection wavelength is 280nm is preferably collected as a CA16 virus purification solution (the two absorption peaks are impurities), and the collection time is preferably 50 to 65min, and more preferably 55 to 60min. The present invention preferably employs an eluent of the kind described above, which can be loaded directly during subsequent purification using a cation exchange chromatography column.
After the CA16 virus purification solution is obtained, the CA16 virus purification solution is inactivated to obtain the CA16 virus inactivation solution. In the present invention, the agent for inactivation is preferably an aqueous formaldehyde solution, and the concentration of the aqueous formaldehyde solution is preferably 24 to 26mg/mL, more preferably 25mg/mL. In the invention, the CA16 virus purified solution is preferably mixed with a formaldehyde aqueous solution for inactivation; the volume ratio of the CA16 virus purification solution to the formaldehyde aqueous solution is preferably based on the condition that the concentration of formaldehyde in the obtained mixed solution is 90-250 mug/mL, and more preferably 90-150 mug/mL; the inactivation temperature is preferably 36.5-37.5 ℃, and more preferably 37.0 ℃; the inactivation time is preferably 7 to 12 days, more preferably 10 to 12 days.
After the CA16 virus inactivation solution is obtained, the CA16 virus inactivation solution is purified by the purification method according to the technical scheme to obtain the CA16 virus inactivation stock solution, and the purification method is not repeated herein.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
(1) Propagation and concentration of CA16 Virus
Inoculating the CA16 virus seeds to KMB17 cells growing into a single layer according to 0.2MOI, and culturing at 37 +/-0.5 ℃ for 48h, wherein the KMB17 cells are completely shrunken to obtain virus liquid; and filtering the virus solution by a 0.45-micron deep filter and a 0.45-micron bacteria-reduction-grade filter in sequence, concentrating by using an ultrafiltration membrane package with the molecular weight cutoff of 100KD, washing the filter membrane of the ultrafiltration membrane package by using 2mmol/L PBS buffer solution, and obtaining virus concentrated solution with the concentration multiple of 60 times.
(2) Molecular sieve chromatographic column purification
Purifying the virus concentrated solution by using a molecular sieve chromatographic column, wherein a molecular sieve in the molecular sieve chromatographic column is Sepharose 6FF, the length of the molecular sieve chromatographic column is 90cm, an equilibrium solution is 2mmol/L PBS buffer solution (pH =7.2 +/-0.2), 3 column volumes are balanced, the sample loading volume is 5% of the column volume, and after sample loading, 1 column volume is continuously eluted by using 2mmol/L PBS buffer solution (pH =7.2 +/-0.2), wherein the linear flow rate during balance, sample loading and elution is 0.255cm/min; and (3) monitoring under the condition that the UV wavelength is 280nm, collecting a sample between two ultraviolet absorption peaks, wherein the collection time is 60min, and obtaining the virus purification solution.
(3) Inactivating
Preparing 25mg/mL formaldehyde aqueous solution, mixing the virus purified solution with the formaldehyde aqueous solution to ensure that the final concentration of formaldehyde is 90 mu g/mL, and then inactivating at 37.0 +/-0.5 ℃ for 12 days to obtain virus inactivated solution.
(4) Cation exchange chromatography column purification
Purifying the virus inactivation solution by using a cation exchange chromatography column, wherein a chromatography medium in the cation exchange chromatography column is POROS TM CEX, wherein the equilibrium solution is 1mmol/L PBS buffer solution (pH =7.2 +/-0.2), 5 column volumes are balanced, the sample loading volume is 3 column volumes, 3 column volumes are continuously balanced after sample loading, and then 0.5mol/L sodium chloride solution is used for elution, wherein the linear flow rates during the balance, the sample loading and the elution are all 1cm/min; monitoring under the condition that the UV wavelength is 280nm, and collecting eluent corresponding to an ultraviolet absorption peak to obtain virus inactivation purified solution (namely virus inactivation stock solution).
Example 2
A purified virus-inactivated solution was prepared in the same manner as in example 1, except that in the cation exchange chromatography column purification step, the eluent used was PBS buffer containing sodium chloride at a concentration of 5mmol/L and 0.5mol/L.
Comparative example 1
The virus-inactivating solution prepared in example 1 was used at 0.1m 2 And concentrating with 100KD ultrafiltration membrane, washing the membrane with 2mmol/L PBS buffer solution, concentrating by 5 times, sterilizing with 0.2 μm filter, and filtering to obtain virus inactivated purified solution.
FIG. 1 is an HPLC chart of the purified virus inactivation solution prepared in comparative example 1, and FIG. 2 is an HPLC chart of the purified virus inactivation solution prepared in example 1; as can be seen from fig. 1 and 2, the purified virus inactivation solution prepared by the method of example 1 of the present invention can remove impurities with retention times of 0.478min and 34.003min from the purified virus inactivation solution prepared by the comparative example 1, and the purity reaches 100%, thereby improving the quality of the purified virus inactivation solution.
The samples obtained in the steps of example 1 and the samples obtained in example 2 and comparative example 1 were analyzed, and the relevant index parameters are specifically shown in table 1. As can be seen from Table 1, the protein removal rate and the antigen recovery rate of the two different eluents used in examples 1-2 of the present invention are significantly improved compared to the method of comparative example 1. Wherein, the removal rate of protein in the virus inactivation purification solution prepared in the embodiment 1 of the invention is 2.3 times of that of the comparative example 1, and the antigen recovery rate in the last step is 1.2 times of that of the comparative example 1, thereby greatly improving the quality of the virus inactivation purification solution. Meanwhile, compared with an ultrafiltration system, the ion exchange chromatographic column purification can realize on-site cleaning, reduce the occupied space of equipment and facilitate the process amplification.
TABLE 1 index parameters of samples obtained in the respective steps of example 1 and samples obtained in example 2 and comparative example 1
Example 3
(1) Propagation and concentration of CA16 Virus
Inoculating the CA16 virus seeds to KMB17 cells growing into a single layer according to 0.15MOI, and culturing at 37 +/-0.5 ℃ for 52h, wherein the KMB17 cells are completely shrunken to obtain virus liquid; and filtering the virus solution by a 0.45-micron deep filter and a 0.45-micron bacteria-reducing filter in sequence, concentrating by using an ultrafiltration membrane package with the molecular weight cutoff of 100KD, washing the filter membrane of the ultrafiltration membrane package by using 2mmol/L PBS buffer solution, and obtaining virus concentrated solution with the concentration multiple of 60 times.
(2) Molecular sieve column purification
Purifying the virus concentrated solution by using a molecular sieve chromatographic column, wherein a molecular sieve in the molecular sieve chromatographic column is Sepharose 6FF, the length of the molecular sieve chromatographic column is 86cm, an equilibrium solution is 2mmol/L PBS buffer solution (pH =7.2 +/-0.2), 3 column volumes are balanced, a sample loading volume is 5% of the column volume, and after sample loading, 1 column volume is continuously eluted by using 2mmol/L PBS buffer solution (pH =7.2 +/-0.2), wherein the linear flow rate in the processes of balancing, sample loading and eluting is 0.255cm/min; and (3) monitoring under the condition that the UV wavelength is 280nm, collecting a sample between two ultraviolet absorption peaks, wherein the collection time is 60min, and obtaining the virus purification solution.
(3) Inactivating
Preparing 25mg/mL formaldehyde aqueous solution, mixing the virus purified solution with the formaldehyde aqueous solution to ensure that the final concentration of formaldehyde is 90 mu g/mL, and then inactivating at 37.0 +/-0.5 ℃ for 12 days to obtain virus inactivated solution.
(4) Cation exchange chromatography column purification
Purifying the virus inactivation solution by using a cation exchange chromatography column, wherein a chromatography medium in the cation exchange chromatography column is SP Bestarose High Performance, an equilibrium solution is 1mmol/L PBS buffer solution (pH =7.2 +/-0.2), 4 column volumes are balanced, a sample loading volume is 3 column volumes, 3 column volumes are continuously balanced after sample loading, and then elution is carried out by using 0.4mol/L sodium chloride solution, wherein the linear flow rate in the processes of balancing, sample loading and elution is 1cm/min; monitoring under the condition that the UV wavelength is 280nm, and collecting eluent corresponding to an ultraviolet absorption peak to obtain virus inactivation purified solution.
Comparative example 2
The virus-inactivating solution prepared in example 3 was used at 0.1m 2 Concentrating with 100KD ultrafiltration membrane package, washing the filter membrane with 2mmol/L PBS buffer solution, concentrating by 5 times, sterilizing with 0.2 μm filter, and filtering to obtain inactivated virusAnd (5) purifying the solution.
The samples obtained in the steps of example 3 and the samples obtained in comparative example 2 were analyzed, and the relevant index parameters are specifically shown in table 2. As can be seen from Table 2, the removal rate of protein in the virus-inactivated purified solution prepared in example 3 of the present invention is 4.5 times that of comparative example 2, and the recovery rate of the antigen in the last step is 1.3 times that of comparative example 2, which greatly improves the quality of the virus-inactivated purified solution.
TABLE 2 index parameters of samples obtained in example 3 and comparative example 2
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for purifying a CA16 virus inactivation solution comprises the following steps:
and purifying the CA16 virus inactivation solution by adopting a cation exchange chromatography column to obtain a CA16 virus inactivation stock solution.
2. The purification method according to claim 1, wherein the exchange packing of the cation exchange chromatography column comprises POROS TM CEX or SP Bestarose High Performance.
3. Purification method according to claim 1 or 2, characterized in that the purification comprises a first equilibration, a first loading, a second equilibration and a first elution, which are carried out in sequence.
4. The purification method according to claim 3, wherein the first equilibration solution is PBS buffer solution, the concentration of the first equilibration solution is 0.8-1.5 mmol/L, and the pH value is 7.0-7.4; the volume of the first equilibrium liquid is 3-5 column volumes.
5. The purification method of claim 3, wherein the volume of the first sample is 1 to 4 column volumes.
6. The purification method according to claim 3, wherein the second equilibrium solution used in the second equilibrium is PBS buffer solution, the concentration of the second equilibrium solution is 0.8-1.5 mmol/L, and the pH value is 7.0-7.4; the volume of the second equilibrium liquid is 1-3 column volumes.
7. The purification method according to claim 3, wherein the eluent used for the first elution is an aqueous sodium chloride solution or a PBS buffer solution containing sodium chloride; the concentration of the sodium chloride aqueous solution is 0.4-1 mol/L; the concentration of the PBS buffer solution in the PBS buffer solution containing sodium chloride is 5-10 mmol/L, and the concentration of the sodium chloride is 0.4-1 mol/L.
8. The purification method according to claim 7, wherein the first elution further comprises ultraviolet detection, and an eluate corresponding to an absorption peak obtained under the condition that the ultraviolet detection wavelength is 280nm is collected as a CA16 virus inactivation stock solution in the first elution process.
9. A preparation method of a CA16 virus inactivation stock solution comprises the following steps:
sequentially proliferating and concentrating the CA16 virus to obtain a CA16 virus concentrated solution;
pre-purifying the CA16 virus concentrated solution by adopting a molecular sieve chromatographic column to obtain a CA16 virus purified solution;
inactivating the CA16 virus purified solution to obtain a CA16 virus inactivated solution;
purifying the CA16 virus inactivation solution by using the purification method of any one of claims 1 to 8 to obtain a CA16 virus inactivation stock solution.
10. The method of claim 9, wherein the pre-purification comprises sequentially performing a third equilibration, a second loading, and a second elution; and the second elution also comprises ultraviolet detection, and eluent between two absorption peaks obtained under the condition that the ultraviolet detection wavelength is 280nm is collected in the second elution process to be used as the CA16 virus purification solution.
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