Method for purifying enterovirus antigen

Abstract

The invention relates to the technical field of biology, in particular to a method for purifying enteroviruses, and more particularly, the method for purifying the enteroviruses comprises the following steps: a) preparing a to-be-purified loading solution containing target enteroviruses; b) combining the loading solution prepared in the step a) with a stationary phase, wherein the enterovirus is immobilized on the stationary phase; c) eluting the enterovirus from the stationary phase with an eluent, thereby obtaining an eluate, the conductance of which is adjusted to gt; 15 mS / cm; and d) optionally further purifying the enterovirus in the eluted collection solution. The method greatly improves the yield of the enterovirus antigen.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for purifying enterovirus antigens. Background Art

[0002] Enterovirus is a class of small, non-enveloped small RNA viruses with single-stranded positive-sense RNA and a single open reading frame. It includes coxsackievirus A, coxsackievirus B, enterovirus EV (enterovirus), echovirus, poliovirus, etc. In terms of structure, mature enterovirus particles are composed of viral capsid proteins VP1, VP2, VP3, VP4 (smaller molecular weight), VP0 and other structural proteins. Enterovirus also has many similar characteristics in terms of pathogenesis, infection and replication characteristics, as well as clinical symptoms and immune responses caused by infection.

[0003] Hand-foot-mouth disease (HFMD) is an acute infectious disease caused by a variety of enterovirus infections. It continues to spread throughout the world, especially in the Asia-Pacific region, posing a serious threat to the lives and health of children. Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the main pathogens causing HFMD, but in recent years, CA6 and CA10 have also become important pathogens of HFMD.

[0004] Currently, the main method of preventing hand, foot and mouth disease is to vaccinate with inactivated vaccines of related viruses. How to efficiently prepare high-purity related virus antigens is the key to vaccine preparation.

[0005] There are relatively few literature reports on detailed methods for the separation and purification of enterovirus. Conventional methods are different combinations of purification methods such as ultrafiltration, centrifugation, ion exchange chromatography, molecular sieve chromatography or multimodal chromatography. Although ion exchange chromatography has a higher yield when using flow-through mode, it has high requirements on the quality of the initial virus harvest liquid. If the quality of the virus harvest liquid is poor, the purity of the virus purification liquid cannot be guaranteed; although the adsorption elution mode has a better impurity removal effect, the recovery rate of viral antigens is unstable.

[0006] Therefore, it is urgent to develop a method for preparing enterovirus antigens that can ensure the purity of viral antigens and have a higher antigen purification yield. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defects in the prior art and provide a method for purifying enterovirus antigens.

[0008] In order to solve the above problems, the technical solution provided by the present invention is as follows:

[0009] A method for purifying enterovirus antigens comprises the following steps:

[0010] a) preparing a sample solution containing target enterovirus antigen to be purified;

[0011] b) combining the sample solution prepared in step a) with a stationary phase, wherein the enterovirus antigen is fixed on the stationary phase; the stationary phase used in step b) is an anion exchange chromatography medium;

[0012] c) using an eluent to elute the enterovirus antigen from the stationary phase to obtain an eluted collection solution, and adjusting the conductivity of the eluted collection solution; wherein adjusting the conductivity of the eluted collection solution in step c) is specifically: adding NaCl, KCl or ammonium sulfate to the eluted collection solution to increase the conductivity of the eluted collection solution to >15mS / cm;

[0013] d) further purifying the enterovirus in the eluted collected fluid; the further purification method in step d) is selected from one or more of ultrafiltration, centrifugation, molecular sieve chromatography, multimodal chromatography, hydrophobic chromatography, and affinity chromatography.

[0014] The term "enterovirus" means that the pathogen that causes hand, foot and mouth disease belongs to the genus Human enterovirus (HEV). According to its serotype classification, it can be mainly divided into four subtypes: HEV-A, HEV-B, HEV-C and HEV-D. HEV-A types include CA2-8, CA10, CA12, CA14, CA16, EV71, EV76, EV89-92; HEV-B types include CA9, CB1-6, E1-7, E9, E11-21, E24-27, E29-33, EV69, EV73, EV74-75, EV77-88, EV93, EV97, EV98, EV100, EV101, EV106, EV107; HEV-C types include CA1, CA11, CA13, CA17, CA19-22, CA24, EV95, EV96, EV99, EV102, EV104, EV105, EV109, PV1-3; HEV-D types include EV68, EV70, EV94. Among them, CA stands for coxsackievirus A; CB stands for coxsackievirus B; EV stands for enterovirus; E stands for echovirus; PV stands for poliovirus.

[0015] In some embodiments, the enterovirus is a human enterovirus that can cause hand, foot and mouth disease, including human enterovirus type A, human enterovirus type B, human enterovirus type C and / or human enterovirus type D classified according to serotype.

[0016] The term "CA16" refers to Coxsackievirus group A type 16, which belongs to type A enterovirus and was isolated by Canadian scientists from patient feces and throat swabs in 1958. However, those skilled in the art should understand that viruses may undergo mutations during culture, propagation or reproduction, so CA16 can be a homologous or heterologous group with no or one or more mutations.

[0017] The term "EV71" refers to enterovirus group A type 71, which belongs to type A enterovirus and was first isolated from the feces of a child with neurological disease in California, USA in 1969. However, those skilled in the art should understand that the virus may undergo mutations during culture, propagation or reproduction, so EV71 can be a homologous or heterologous group with no or one or more mutations.

[0018] In some embodiments, the enterovirus is a human enterovirus type A. In some embodiments, the enterovirus includes CA16, CA6, CA10, EV71.

[0019] The complete life cycle of enterovirus includes virus adsorption, uncoating, replication, transcription and translation of viral genome, assembly of viral capsid, virus maturation and virus release. The term "mature enterovirus" refers to the virus assembled from the host cell and the virus released from the host cell. Mature enterovirus is composed of 5 kinds of viral coat proteins, namely VP0, VP1, VP2, VP3, and VP4 (smaller molecular weight). In some embodiments, there are both infectious mature virus particles and non-infectious hollow virus particles in the virus harvest liquid, both of which are effective antigen components of the vaccine. Preferably, the enterovirus is a mature enterovirus or a hollow particle without nucleic acid, and its coat protein includes at least structural proteins VP1, VP2, and VP3.

[0020] The term "immobilization" refers to the combination of enterovirus with the immobilization agent through interactions, including electrostatic interactions of anions and cations, solubility distribution, ion exchange, and exclusion.

[0021] The term "stationary phase" refers to any surface on which groups that can interact with the target sample can be fixed. The stationary phase can be a suspension, a purification column, a discontinuous phase of discrete particles, a plate, a sensor, a chip, a capsule, a resin, a gel, a membrane, a filter, etc.; the group can be one or more charged groups, and commonly used materials that form a stationary phase with charged groups include organic copolymer resins, silica microspheres, chelating resins, etc.;

[0022] The term "ion exchange chromatography" refers to the stationary phase in ion exchange chromatography that contains some charged groups on its surface, such as SO3 - 、COO - , NH3+, etc. These charged groups bind to ions and / or proteins with opposite charges in the mobile phase (including target sample ions, impurity ions, etc.) through electrostatic interactions, and control the competitive effect of target sample ions and / or proteins with impurity ions and / or proteins on the surface charge of the stationary phase, so as to separate the target sample ions and / or proteins from the impurities. From the perspective of group charge, ion exchange chromatography includes cation exchange chromatography and anion exchange chromatography.

[0023] In some embodiments, in step b), the enterovirus binds to the stationary phase through an interaction selected from one of adsorption, partitioning, ion exchange or exclusion. In some embodiments, the stationary phase used in step b) is an anion exchange chromatography medium, which can adsorb enterovirus antigen particles, thereby fixing the enterovirus antigen particles on the medium. In some embodiments, the anion exchange medium used in step b) is selected from one or more of Capto Q, CaptoDEAE or Source Q.

[0024] The term "equilibrium solution" refers to a solution that equilibrates the stationary phase before loading the solution containing the enterovirus antigen onto the stationary phase, the purpose of which is to balance the stationary phase with the initial environment, and when equilibrium is reached, all charged groups on the stationary phase are bound to exchangeable ions and / or proteins with opposite charges. The equilibrium solution is one or more of a salt and a buffer. In some embodiments, the equilibrium solution is a PB buffer or a Tris-HCl buffer that contains no or only a low concentration of a salt such as NaCl, KCl or ammonium sulfate.

[0025] The term "loading solution" refers to a solution used to load the composition comprising the target enterovirus antigen and one or more impurities onto the stationary phase. The loading solution may optionally include a buffer.

[0026] In some embodiments, the loading solution of step a) is a PB buffer or a Tris-HCl buffer containing the target enterovirus antigen, and the loading solution contains a salt solution such as 10-150 mmol / L NaCl, KCl or ammonium sulfate. The method for preparing the loading solution of step a) is selected from one or more of deep filtration, ultrafiltration, centrifugation, chromatography, and clarification filtration.

[0027] The term "elution solution" refers to a solution used to elute the target enterovirus antigen from the stationary phase. The elution solution may contain one or more of a salt and a buffer.

[0028] In some embodiments, the salt ions in the eluent of step c) competitively bind to the stationary phase, thereby eluting the enterovirus antigen particles from the stationary phase and following the eluent into the eluent collection solution. In some embodiments, the conductivity of the eluent is higher than that of the loading solution and the balancing solution. In some embodiments, the eluent is a PB buffer or a Tris-HCl buffer containing 155 mmol / L of NaCl, KCl or ammonium sulfate.

[0029] The term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, electric current flows by the transport of ions. Therefore, as the amount of ions present in an aqueous solution increases, the solution will have a higher conductivity. Conductivity is measured by a conductivity meter. The conductivity of a solution can be changed by changing the concentration of ions in the solution, for example, the concentration of a buffer and / or salt in the solution can be changed to achieve a desired conductivity.

[0030] In some embodiments, a high concentration of NaCl, KCl or ammonium sulfate is added to the elution collection solution in step c) to increase the conductivity of the elution collection solution to >15 mS / cm. In some embodiments, the elution collection solution with a conductivity of >15 mS / cm enhances the hydrophobic effect of the viral capsid antigen and increases the stability of the viral particles.

[0031] In some embodiments, step i) may be further included between step b) and step c) to wash away substances not bound to the stationary phase.

[0032] In some embodiments, the pH value of one or more of the loading solution, the equilibration solution, and the eluent is between pH 6-9.

[0033] In some embodiments, the optional other purification methods in step d) are selected from one or more of ultrafiltration, centrifugation, molecular sieve chromatography, multimodal chromatography, hydrophobic chromatography, and affinity chromatography.

[0034] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virus, protein, antigen, microbiology, purification-related terms and laboratory operation procedures used in the present invention are terms and conventional procedures widely used in the corresponding fields.

[0035] As used herein, the term "one or more selected from..." encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "one or more selected from A, B, C" encompasses "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0036] "Purification" of the intended enteroviral antigen refers to increasing the purity of the enteroviral antigen in the composition by removing (completely or partially) at least one impurity from the composition. Impurities may be host cell components and their metabolites such as host proteins or nucleic acids, cell debris, cell or viral culture media, etc. The term is not intended to refer to the complete absence of such biomolecules or the absence of water, buffers or salts or the absence of components of the pharmaceutical formulation comprising the enteroviral antigen.

[0037] The beneficial effect of the present invention is that the present invention has developed a method for purifying enterovirus antigens based on anion exchange chromatography adsorption-elution mode. At present, there is no report on the control of conductivity for enterovirus collection samples to improve the purification yield and purity of viral antigens. The inventors of the present application have found that when the adsorption-elution mode is used to purify viral antigens, the sharp drop in conductivity is the key factor causing the instability of viral antigens due to the use of low-salt loading and high-salt elution. In order to reduce the adverse effects of the low-conductivity environment on the stability of viral particles during the adsorption-elution process, the inventors timely adjust the conductivity of the elution collection liquid of the anion exchange chromatography, and maintain the strong hydrophobic effect of the viral capsid antigen in a high-salt environment to maintain the stability of the viral particle structure, reduce the lysis loss of viral particles in the subsequent purification process, and thus effectively improve the antigen recovery rate of the entire purification process.

[0038] In some embodiments of the present invention, using the technical solution provided by the present invention, the total protein yield of enterovirus CA16 after purification is increased from 0.43 μg / mL when the conductivity is not adjusted to 1.08 μg / mL, the total antigen yield is increased from 0.13 μg / mL to 1.36 μg / mL, and the total specific activity is increased from 0.29 μg / μg to 1.27 μg / μg; the total protein yield of enterovirus EV71 after purification is increased from 0.19 μg / mL when the conductivity is not adjusted to 1.06 μg / mL, the total antigen yield is increased from 78.10 U / mL to 1370.56 U / mL, and the total specific activity is increased from 412.58 U / μg to 1288.36 U / μg. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 .CA16-P006 anion exchange chromatography - adsorption elution mode chromatogram (without increasing the conductivity of the elution collection solution).

[0040] Figure 2 .CA16-P006 molecular sieve chromatography (without increasing the conductivity of the elution collection solution).

[0041] Figure 3 .CA16-P006 SDS-PAGE gel image (without increasing the conductivity of the elution collection solution).

[0042] Figure 4 .CA16-T022 anion exchange chromatography-adsorption elution mode chromatogram (increasing the conductivity of the elution collection liquid), in which the two peaks at 4750-5000mL are the target products.

[0043] Figure 5 .CA16-T022 molecular sieve chromatography (increasing the conductivity of the elution collection solution), where peak 1 is the target product.

[0044] Figure 6 .CA16-T022 SDS-PAGE gel image (increasing the conductivity of the elution collection solution).

[0045] Figure 7 .CA16-P012 anion exchange chromatography-adsorption elution mode chromatogram, where the peak between 1800-2300mL (peak 2) is the target product (increasing the conductivity of the elution collection solution).

[0046] Figure 8 .CA16-P012 molecular sieve chromatography spectrum, where the peak between 0.50-0.60CV (peak 1) is the target product (increasing the conductivity of the elution collection solution).

[0047] Fig. 9 .CA16-P012 SDS-PAGE gel image (increasing the conductivity of the elution collection solution).

[0048] Fig.10 .EV71-P012 anion exchange chromatography-adsorption elution mode chromatogram, where the peak between 2100-2300mL (peak 2) is the target product (without increasing the conductivity of the elution collection solution).

[0049] Fig.11 .EV71-P012 molecular sieve chromatography spectrum, where the peak between 0.50-0.60CV (peak 1) is the target product (without increasing the conductivity of the elution collection solution).

[0050] Fig.12 .EV71-P012 SDS-PAGE gel image (without increasing the conductivity of the elution collection solution).

[0051] Fig.13 .EV71-P013 anion exchange chromatography-adsorption elution mode chromatogram, where the peak between 1900-2100mL is the target product (increasing the conductivity of the elution collection liquid).

[0052] Fig.14 .EV71-P013 molecular sieve chromatography spectrum, where the peak between 0.40-0.53CV (peak 1) is the target product (increasing the conductivity of the elution collection solution).

[0053] Fig.15 .EV71-P013 SDS-PAGE gel image (increasing the conductivity of the elution collection solution). DETAILED DESCRIPTION

[0054] The present invention is illustrated below with reference to examples, but the present invention is not limited thereto. In the art, simple replacements or improvements made to the present invention by technicians are all within the technical solution protected by the present invention. Obviously, those skilled in the art know that various modifications and changes can be made to the present invention without departing from the spirit of the present invention, so such modifications and changes also fall within the scope of the present invention. In order to illustrate the technical solution and technical effects of the present invention, the present invention selects common enterovirus CA16 and EV71, but those skilled in the art know that other enteroviruses such as CA6 and CA10 are also applicable to the technical solution of the present invention.

[0055] Example 1: Purification of enterovirus CA16 antigen using conventional purification method (without increasing the conductivity of the eluted collection solution)

[0056] 1.1 Preparation of the sample solution containing the target viral antigen to be purified by chromatography

[0057] After the CA16 virus culture fluid is harvested, in order to prepare a sample solution suitable for ion exchange chromatography, it is necessary to remove impurities such as cell debris in the culture fluid. Therefore, the cultured CA16 virus harvest fluid is first filtered using a Cobetter Claricap L deep filter, and rinsed with an appropriate amount of PB buffer containing 150mmol / L NaCl, and combined into a deep filtrate. Then, the deep filtrate is concentrated 20 times (based on the volume of the initial virus harvest fluid) using a Cobetter 100kD membrane package, washed 10 times the volume and replaced with a PB buffer solution without NaCl to obtain an ultrafiltration product. After measurement, its conductivity is 2.1mS / cm. The ultrafiltration product will be directly loaded as a sample solution in the next step of ion exchange chromatography.

[0058] 1.2 Adsorb and bind the enterovirus antigen to the stationary phase and then elute to obtain the elution collection solution

[0059] Anion exchange chromatography was performed using a Cypress S100 chromatograph. After the Capto Q column was equilibrated with a pH 7.2 PB buffer without NaCl, the sample prepared in step 1.1 was loaded onto the Capto Q column. Enterovirus in the column was eluted with a PB buffer containing 155 mmol / L NaCl and pH 7.2, and the eluate under the target peak was collected to obtain an elution collection solution (Q eluate), the conductivity of which was 11 mS / cm. The ion exchange chromatogram is shown in FIG. Figure 1 1.3 Further purification after chromatography

[0060] Generally, in order to meet the purity requirements of vaccine products, other purification steps such as multimodal chromatography and molecular sieve chromatography need to be superimposed after ion exchange chromatography. The present invention uses molecular sieve chromatography after ion exchange chromatography as an example for further purification.

[0061] In order to inactivate the virus, formaldehyde solution was first added to the Q eluate to a final formaldehyde concentration of 250 μg / mL, and the virus was inactivated at 37°C for 66 h to obtain a formaldehyde inactivated solution. Then, in order to remove formaldehyde and prepare a molecular sieve chromatography loading solution, the formaldehyde inactivated solution was concentrated to about 7-10 mL using a Cobite 100 kD membrane package, and the solution was washed and filtered 10 times the volume and replaced with a PB buffer containing 150 mmol / L NaCl to obtain an inactivated concentrated solution, which was also the molecular sieve chromatography loading solution.

[0062] Molecular sieve chromatography was performed using a Sephacryl S100 chromatograph. The Sephacryl S300 column was equilibrated with PB buffer containing 150 mmol / L NaCl, and then <5% of the column volume of the inactivated concentrate was loaded and peak 1 was collected. The chromatogram is shown in Figure 2 The sample was filtered through 0.22 μm to obtain the purified virus solution.

[0063] 1.4 Analysis of purification results

[0064] The protein content was tested according to the Lowry method specified in the second method of General Chapter 0731 of the 2020 edition of the Chinese Pharmacopoeia;

[0065] The sample purity was tested according to the SDS-PAGE method specified in the fifth method of General Chapter 0541 of the 2020 edition of the Chinese Pharmacopoeia;

[0066] The Elisa method for detecting antigen content is as follows: Coat the ELISA plate with CA16 rabbit polyclonal antibody, add samples and standards diluted to appropriate concentrations, set up negative control wells, and wash the plate after incubation. Add horseradish peroxidase-labeled CA16 monoclonal antibody, wash the plate after incubation, add color developer, and measure the absorbance value with an ELISA reader after color development. Use the standard concentration as the X-axis and the Log value of the absorbance value as the Y-axis for four-parameter fitting, and calculate the sample concentration based on the sample absorbance value;

[0067] The results of protein content and antigen content detection in each step of the purification process are summarized in Table 1. It can be seen from Table 1 that the antigen recovery rate drops sharply after ultrafiltration 2. The results of SDS-PAGE sample purity detection are shown in Figure 3 As shown, from Figure 3 It can be seen that the antigen content of enterovirus CA16 in the final purified solution is low.

[0068] The calculation or statistical method of protein removal rate is: (1-total amount of harvested protein / total amount of loaded protein)*100%

[0069] The calculation or statistical method of antigen recovery rate is: total amount of harvested antigen / total amount of loaded antigen*100%

[0070] The calculation or statistical method of specific activity is: total antigen amount / total protein amount

[0071] Table 1. CA16 protein and antigen amounts obtained in each step using conventional purification methods

[0072]

[0073]

[0074] Example 2: Improving the Purification Yield of Enterovirus CA16 Antigen by Improving the Conductivity of the Elution Collection Solution

[0075] 2.1 Preparation of the sample solution containing the target viral antigen to be purified by chromatography

[0076] After the CA16 virus culture fluid is harvested, in order to prepare a sample solution suitable for ion exchange chromatography, it is necessary to remove impurities such as cell debris in the culture fluid. Therefore, the cultured CA16 virus harvest fluid is first filtered using a Cobetter Claricap L deep filter, and rinsed with an appropriate amount of PB buffer containing 150mmol / L NaCl, and combined into a deep filtrate. After concentrating 20 times (based on the initial virus harvest fluid volume) using a Cobetter 100kD membrane package, wash the 10-fold volume and change the solution to a PB buffer containing 150mmol / L NaCl. The ultrafiltration product is diluted 5 times with a pH 7.2 PB buffer without NaCl as the sample solution, and the conductivity of the sample solution is 5.3mS / cm.

[0077] 2.2 Adsorbing the enterovirus antigen to the stationary phase and then eluting to obtain the elution collection solution

[0078] Anion exchange chromatography was performed using a Cypress S100 chromatograph. The Capto Q column was equilibrated with a PB buffer containing no NaCl and pH 7.2. The sample solution prepared in step 2.1 was loaded onto the Capto Q column. CA16 adsorbed on the column was eluted with a PB buffer containing 155 mmol / L NaCl and pH 7.2, and the eluate under the target peak was collected to obtain an eluted collection solution (Q eluate). The conductivity of the eluted collection solution was 8.7 mS / cm. The ion exchange chromatogram is shown in FIG. Figure 4 shown.

[0079] 2.3 Improve the conductivity of the collected eluent

[0080] After obtaining the elution collection solution in step 2.2, immediately adjust the conductivity of the elution collection target sample to 19 mS / cm with 4 M NaCl solution.

[0081] 2.4 Further purification after chromatography is completed is the same as step 1.3 in Example 1. The molecular sieve chromatography spectrum is as follows: Figure 5 shown.

[0082] 2.5 Analysis of purification results The same as step 1.4 in Example 1, the SDS-PAGE sample purity test results are as follows: Figure 6 shown.

[0083] The summary results of the CA16 protein content and antigen content detection in each step of the purification process are shown in Table 2. It can be seen from Table 2 that after anion exchange chromatography, by increasing the conductivity of the elution collection solution, the antigen recovery rate in the subsequent steps is significantly increased. The antigen recovery rate of ultrafiltration 2 increased from 15.1% before the conductivity was not increased to 98.7%.

[0084] The results of SDS-PAGE sample purity test are as follows Figure 6 As shown, from Figure 6 It can be seen that the antigen content of enterovirus CA16 in the final purified solution is significantly increased.

[0085] Table 2. CA16 protein and antigen amounts obtained through each step under the condition of increasing the conductivity of the elution collection solution

[0086]

[0087]

[0088] Example 3: Improving the Purification Yield of Enterovirus CA16 Antigen by Improving the Conductivity of the Elution Collection Solution

[0089] 3.1 Preparation of the sample solution containing the target viral antigen to be purified by chromatography

[0090] After the CA16 virus culture fluid is harvested, in order to prepare a sample solution suitable for ion exchange chromatography, it is necessary to remove impurities such as cell debris in the culture fluid. Therefore, the cultured CA16 virus harvest fluid is first filtered using a Cobetter Claricap L deep filter, and rinsed with an appropriate amount of PB buffer containing 150mmol / L NaCl, and combined into a deep filtrate. After concentrating 20 times (based on the initial virus harvest fluid volume) using a Cobetter 100kD membrane package, wash the 10-fold volume and change the solution to PB buffer containing 150mmol / L NaCl. The ultrafiltration product is diluted 5 times with a pH 7.2 PB buffer without NaCl as the sample solution, and the conductivity of the sample solution is 5.0mS / cm.

[0091] 3.2 Adsorbing the enterovirus antigen to the stationary phase and then eluting to obtain the elution collection solution

[0092] Anion exchange chromatography was performed using a Cypress S100 chromatograph. The Capto Q column was equilibrated with a PB buffer containing no NaCl and pH 7.2. The sample solution prepared in step 3.1 was loaded onto the Capto Q column. CA16 adsorbed on the column was eluted with a PB buffer containing 155 mmol / L NaCl and pH 7.2, and the eluate under the target peak was collected to obtain the eluted collection solution (Q eluate). The conductivity of the eluted collection solution was 8.4 mS / cm. The ion exchange chromatogram is shown in FIG. Figure 7 shown.

[0093] 3.3 Improve the conductivity of the collected eluent

[0094] After obtaining the elution collection solution in step 3.2, immediately adjust the conductivity of the elution collection target sample to 30 mS / cm with 4 M NaCl solution.

[0095] 3.4 Further purification after chromatography is completed is the same as step 1.3 in Example 1. The molecular sieve chromatography spectrum is as follows: Figure 8 3.5 Analysis of purification results The same as step 1.4 in Example 1, the SDS-PAGE sample purity test results are shown as follows Fig. 9 shown.

[0096] The summary results of the CA16 protein content and antigen content detection in each step of the purification process are shown in Table 3. It can be seen from Table 3 that after anion exchange chromatography, the conductivity of the eluted collection solution was further increased to 30mS / cm, and the antigen recovery rate in the subsequent steps was still improved. The antigen recovery rate of ultrafiltration 2 increased from 15.1% before the conductivity was not increased to 62.2%.

[0097] The results of SDS-PAGE sample purity test are as follows Fig. 9 As shown, from Fig. 9 It can be seen that the antigen content of enterovirus CA16 in the final purified solution is significantly increased.

[0098] Table 3. CA16 protein and antigen amounts obtained through each step under the condition of increasing the conductivity of the elution collection solution

[0099]

[0100] Example 4: Purification of enterovirus EV71 antigen using conventional purification method (without increasing the conductivity of the eluted collection solution)

[0101] 4.1 Preparation of the sample solution containing the target viral antigen to be purified by chromatography

[0102] After harvesting the EV71 virus culture fluid, in order to prepare a sample solution suitable for ion exchange chromatography, it is necessary to remove impurities such as cell debris in the culture fluid. Therefore, the cultured EV71 virus harvest fluid is first filtered using a Cobetter Claricap L deep filter, and rinsed with an appropriate amount of PB buffer containing 150mmol / L NaCl, and combined into a deep filtrate. Then, after concentrating 20 times (based on the volume of the initial virus harvest fluid) using a Cobetter 100kD membrane package, the 10-fold volume is washed and replaced with a PB buffer solution without NaCl to obtain an ultrafiltration product. The conductivity was measured to be 2.9mS / cm. The ultrafiltration product will be directly loaded as a sample solution in the next step of ion exchange chromatography.

[0103] 4.2 Adsorbing the enterovirus antigen to the stationary phase and then eluting to obtain the elution collection solution

[0104] Anion exchange chromatography was performed using an Insex Unique AutoPure25 chromatograph. The Capto Q column was equilibrated with PB buffer at pH 7.2 without NaCl. The sample solution in step 4.1 was loaded onto the Capto Q column. Enterovirus in the column was eluted with PB buffer at pH 7.2 containing 155 mmol / L NaCl, and the eluate under the target peak was collected to obtain the eluted collection solution (Q eluate). The conductivity of the eluted collection solution was 12.7 mS / cm. The ion exchange chromatogram is shown in FIG. Fig.10 shown.

[0105] 4.3 Further purification after chromatography

[0106] Generally, in order to meet the purity requirements of vaccine products, other purification steps such as multimodal chromatography and molecular sieve chromatography need to be superimposed after ion exchange chromatography. The present invention uses molecular sieve chromatography after ion exchange chromatography as an example for further purification.

[0107] In order to inactivate the virus, firstly, formaldehyde mother solution was added to the Q eluate to make the final formaldehyde concentration 250 μg / mL, and inactivated at 37°C for 66 h to obtain formaldehyde inactivated solution. Then, in order to remove formaldehyde and prepare the molecular sieve chromatography loading solution, the formaldehyde inactivated solution was concentrated to about 10 mL using a Cobite 100 kD membrane package, and then washed and filtered 10 times the volume and replaced with a PB buffer containing 150 mmol / L NaCl to obtain an inactivated concentrated solution, which is also the molecular sieve chromatography loading solution.

[0108] Molecular sieve chromatography was performed using a Sephacryl S100 chromatograph. The Sephacryl S300 column was equilibrated with PB buffer containing 150 mmol / L NaCl, and then <5% of the column volume of the inactivated concentrate was loaded and peak 1 was collected. The molecular sieve chromatography chromatogram is shown in Fig.11 After 0.22 μm filtration, the virus purification solution was obtained.

[0109] 4.4 Analysis of purification results

[0110] The protein concentration was tested according to the Lowry method specified in the second method of General Chapter 0731 of the 2020 edition of the Chinese Pharmacopoeia;

[0111] The sample purity was tested according to the SDS-PAGE method specified in the fifth method of General Chapter 0541 of the 2020 edition of the Chinese Pharmacopoeia;

[0112] Elisa method for detecting antigen content is as follows: coat the ELISA plate with EV71 rabbit polyclonal antibody, add samples and standards diluted to appropriate concentrations, set up negative control wells, incubate and wash the plate. Add horseradish peroxidase-labeled EV71 monoclonal antibody, incubate and wash the plate, add color developer, and measure the absorbance with an ELISA reader after color development. Use the standard concentration as the X-axis and the Log value of the absorbance as the Y-axis for four-parameter fitting, and calculate the sample concentration based on the sample absorbance.

[0113] The results of protein content and antigen content detection in each step of the purification process are summarized in Table 4. It can be seen from Table 4 that the antigen recovery rate drops sharply after anion exchange chromatography. Fig.12 As shown, from Fig.12 It can be seen that the antigen content of enterovirus EV71 in the final purified liquid is low.

[0114] Table 4. EV71 protein and antigen amounts obtained in each step using conventional purification methods

[0115]

[0116]

[0117] Example 5: Improving the Purification Yield of Enterovirus EV71 Antigen by Improving the Conductivity of the Elution Collection Solution

[0118] 5.1 Preparation of the sample solution containing the target viral antigen to be purified by chromatography

[0119] After harvesting the EV71 virus culture fluid, in order to prepare a sample solution suitable for ion exchange chromatography, it is necessary to remove impurities such as cell debris in the culture fluid. Therefore, the cultured EV71 virus harvest fluid was first filtered using a Cobetter Claricap L depth filter, and rinsed with an appropriate amount of PB buffer containing 150mmol / L NaCl, and combined into a deep filtrate. After concentrating 20 times (based on the initial virus harvest fluid volume) using a Cobetter 100kD membrane package, the 10-fold volume was washed and replaced with a PB buffer containing 150mmol / L NaCl. The ultrafiltration product was diluted 5 times with a pH 7.2 PB buffer without NaCl as the sample solution, and the conductivity of the sample solution was 5.9mS / cm.

[0120] 5.2 Adsorbing the enterovirus antigen to the stationary phase and then eluting to obtain the elution collection solution

[0121] Anion exchange chromatography was performed using a Cypress S100 chromatograph. The Capto Q column was equilibrated with a PB buffer containing no NaCl and pH 7.2. The sample solution prepared in step 5.1 was loaded onto the Capto Q column. The EV71 adsorbed on the column was eluted with a PB buffer containing 155 mmol / L NaCl and pH 7.2, and the eluate under the target peak was collected to obtain the eluted collection solution (Q eluate). The conductivity of the eluted collection solution was 14 mS / cm. The ion exchange chromatogram is shown in FIG. Fig.13 shown.

[0122] 5.3 Improve the conductivity of the collected eluent

[0123] After obtaining the elution collection solution in step 5.2, immediately adjust the conductivity of the elution collection target sample to 17 mS / cm with 4 M NaCl solution.

[0124] 5.4 Further purification after chromatography is completed is the same as step 4.3 in Example 4. The molecular sieve chromatography spectrum is as follows: Fig.14 5.5 Analysis of purification results The same as step 4.4 in Example 4, the SDS-PAGE sample purity test results are shown as follows Fig.15 shown.

[0125] The summary results of the EV71 protein content and antigen content detection in each step of the purification process are shown in Table 5. It can be seen from Table 5 that after anion exchange chromatography, the conductivity of the elution collection solution was timely improved, and the protein and antigen recovery rates of the subsequent purification steps were significantly improved. For example, the antigen recovery rate after molecular sieve chromatography increased from 18.3% without increasing the conductivity to 97.3%.

[0126] Table 5. EV71 protein and antigen amounts obtained through each step under the condition of increasing the conductivity of the elution collection solution

[0127]

[0128]

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for purifying enterovirus antigens, characterized in that: The steps include: a) preparing a sample solution containing target enterovirus antigen to be purified; b) combining the sample solution prepared in step a) with a stationary phase, wherein the enterovirus antigen is fixed on the stationary phase; the stationary phase used in step b) is an anion exchange chromatography medium; c) using an eluent to elute the enterovirus antigen from the stationary phase to obtain an eluted collection solution, and adjusting the conductivity of the eluted collection solution; wherein adjusting the conductivity of the eluted collection solution in step c) is specifically: adding NaCl, KCl or ammonium sulfate to the eluted collection solution to increase the conductivity of the eluted collection solution to >15mS / cm; d) further purifying the enterovirus in the eluted collected fluid; the further purification method in step d) is selected from one or more of ultrafiltration, centrifugation, molecular sieve chromatography, multimodal chromatography, hydrophobic chromatography, and affinity chromatography.

2. The method according to claim 1, characterized in that Before the step a), the stationary phase is balanced with a balancing solution, wherein the balancing solution is a PB buffer or a Tris-HCl buffer that does not contain NaCl, KCl or ammonium sulfate.

3. The method according to claim 1, characterized in that The method for preparing the sample solution in step a) is selected from one or more of deep filtration, ultrafiltration, centrifugation, chromatography, and clarification filtration.

4. The method according to claim 1, characterized in that: The loading solution in step a) is a PB buffer or a Tris-HCl buffer containing the target enterovirus, and the loading solution contains 10-150 mmol / L NaCl, KCl or ammonium sulfate.

5. The method according to claim 1, characterized in that The anion exchange chromatography medium used in step b) is selected from one or more of Capto Q, Capto DEAE or Source Q.

6. The method according to any one of claims 1 to 5, characterized in that: The eluent was PB buffer or Tris-HCl buffer containing 155 mmol / L NaCl, KCl or ammonium sulfate.

7. The method according to any one of claims 2 to 5, characterized in that: The pH values ​​of the loading solution, the balancing solution and the eluting solution are between 6 and 9, and the conductivity of the eluting solution is higher than that of the loading solution and the balancing solution.

8. The method according to claim 1, characterized in that: The method further comprises step i) washing away substances not bound to the stationary phase between step b) and step c).

9. The method according to claim 1, characterized in that: The enterovirus is a human enterovirus that can cause hand, foot and mouth disease, including human enterovirus type A, human enterovirus type B, human enterovirus type C and / or human enterovirus type D classified according to serotype.

10. The method according to claim 9, characterized in that The enterovirus is human enterovirus type A.

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