Preparation method and application of oncolytic adenovirus for improving purification and infection efficiency

Abstract

The invention belongs to the technical field of biopharmaceutical processes and biological medicines, and particularly relates to a preparation method and application of oncolytic adenovirus capable of improving purification and infection efficiency. Aiming at the problems of high purification cost, high loss rate and low oncolytic efficiency of the existing oncolytic adenovirus, the invention provides the preparation method of the oncolytic adenovirus, and on the basis of the existing three-step method for separating and purifying the oncolytic adenovirus, desalination chromatography is reduced, and the purification cost is reduced. An existing three-step method for purifying the oncolytic adenovirus is improved and reduced into a two-step method, so that the purification time of the virus is greatly shortened, the loss of the virus is reduced, and the recovery rate of the virus is increased. In addition, the prepared oncolytic adenovirus is higher in replication efficiency in tumors and better in anti-tumor effect. The method for producing the oncolytic adenovirus is simpler, more economical, more efficient, more concise and suitable for industrial production, the cost is reduced, the yield of the oncolytic adenovirus is improved, and the method has a very good utilization prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology and biomedicine technology, and specifically relates to a method for preparing an oncolytic adenovirus with improved purification and infection efficiency and its use; more specifically, the present invention optimizes the original process for separating and purifying oncolytic adenoviruses to produce oncolytic adenoviruses and improve the oncolytic effect of oncolytic adenoviruses. Background Art

[0002] With the advancement and popularization of medical examination methods, the incidence of cancer has increased year by year in recent years. It has now become the second leading cause of death in the world, causing a heavy economic burden on ordinary people and the public health system. Traditional treatments for cancer include surgery, chemotherapy, and radiotherapy. However, traditional radiotherapy and chemotherapy have large side effects, poor patient response rate, and are prone to drug resistance. In recent years, targeted drugs targeting specific driver gene targets and immune checkpoint inhibitors represented by PD-1 / PD-L1 antibodies have gradually emerged, greatly improving the prognosis of some cancer patients, but only a few patients can benefit from them. Therefore, we need to find a more broad-spectrum, efficient, and lasting treatment method. With the rapid development of disciplines such as molecular biology, cell biology, and immunology, and with the continuous updating and iteration of genetic engineering technology, biological treatments represented by gene therapy and tumor immunotherapy have gradually moved from laboratory research to clinical exploration, which is expected to break the current dilemma of cancer treatment.

[0003] Gene therapy refers to a new biomedical technology that introduces exogenous normal genes and therapeutic genes, such as immune targets, cytokines, etc., into target cells in a certain way to correct abnormal genes or compensate for defective genes, thereby achieving the purpose of treating corresponding diseases. The emergence of gene therapy has brought new treatment options for tumors, genetic diseases, autoimmune diseases, etc. The vectors of gene therapy are divided into non-viral vectors and viral vectors. Non-viral vectors use the physicochemical properties of non-viral vector materials to mediate gene transfer. Non-viral vectors mainly include liposome complexes, cationic polymers (polylysine, polyethyleneimine, dendritic polymers, etc.), chitosan polymers and inorganic nanoparticles. In recent years, plasmids or naked DNA have begun to attract attention as a new therapeutic non-viral vector. The transfection efficiency of non-viral vectors is low, which is mainly because non-viral vector-mediated gene therapy needs to overcome three barriers: cell membrane, endosomal-lysosome system, and nuclear membrane. The target gene carried by it can only be expressed transiently, and it is very easy to cause immune response, resulting in self-clearance by the body, so it is difficult to achieve the effect of treating diseases. Viral vectors can carry genetic material into a variety of cells (tumor cells, fibroblasts, T cells, B cells, macrophages, NK cells, etc.) through infection, thereby changing the functional state of the above cells, and then improving the tumor microenvironment, and ultimately achieving the purpose of treating tumors. Common viral vectors include adenoviruses, adeno-associated viruses, lentiviruses, and retroviruses. The transduction efficiency of viral vectors is very high, and the complex assembly process is completed by cells. Different viral vectors have different expression characteristics. Oncolytic adenovirus is a viral vector modified from adenovirus that is replicable specifically in tumor cells.

[0004] Oncolytic adenoviruses (OVs) can be modified through molecular biological methods to replicate specifically in tumor cells and cause tumor cell lysis, ultimately leading to tumor cell rupture and death, with no effect on normal cells, thereby activating the immune system to generate an immune response. This type of virus is generally believed to exert its anti-tumor effect in the body through two mechanisms: first, OVs replicate specifically in tumor cells, leading to direct tumor cell lysis; second, the anti-tumor response of OVs in tumor cells can produce a large number of tumor antigens, which, through antigen presentation, prompt immune cells to release a large number of immunomodulatory factors, promoting the body's systemic anti-tumor response. As a new method for treating tumors, OVs are widely used in clinical and preclinical research.

[0005] In order to meet the needs of clinical trials, large-scale production of oncolytic adenovirus has become a key issue of our concern. At present, the main method for producing oncolytic adenovirus in the laboratory is CsCl density gradient ultracentrifugation, but the yield of oncolytic adenovirus obtained by this method is limited and can only be applied to preclinical research scenarios, and cannot meet the needs of large-scale clinical applications; with the development and application of chromatography systems, the methods for producing oncolytic adenovirus have also developed to a certain extent. In recent years, the chromatography method for producing oncolytic adenovirus is mainly based on the negatively charged characteristics of the virus, and different combinations of purification systems are selected to separate and purify the oncolytic adenovirus; the current methods for chromatographic separation and purification of oncolytic adenovirus are: 1. Combination of weak anion, strong anion and desalting chromatography; 2. Combination of weak anion and molecular sieve; 3. Mixed filler combining anion and molecular sieve (Capto TM core700), desalting chromatography and other methods were used to separate and purify oncolytic adenovirus.

[0006] According to the above-mentioned methods for separating and purifying oncolytic adenoviruses, there are many steps, the loss rate of virus in each purification process is large, and the final recovered oncolytic adenovirus is small, and the recovery rate accounts for about 20-30% of the fermentation output. The obtained oncolytic adenovirus has low oncolytic efficiency and takes a long time, which is not suitable for use in large-scale clinical production. Therefore, we urgently need to research and develop a purification method that is simple to operate, low in cost, high in lysis recovery rate, high in oncolytic efficiency, and short in time. Summary of the invention

[0007] The technical problem to be solved by the present invention is that the existing method for producing oncolytic adenovirus is costly, the virus loss is large during the purification process, and the recovery rate is low; the oncolytic adenovirus product obtained has low oncolytic efficiency and poor efficiency in treating tumors.

[0008] The technical solution of the present invention to solve the above technical problems is: to provide a method for preparing oncolytic adenovirus with improved purification and infection efficiency. The method comprises the following steps:

[0009] a. Resuscitate HEK293 cells and incubate at 37±1°C, 5±0.5% CO 2 Cultivated under conditions;

[0010] b. Prepare new DMEM culture medium, add oncolytic adenovirus seed solution according to the multiplicity of infection of 20-30, and the cells cultured in step a, and incubate at 37±1°C, 5±0.5% CO 2 Conditions were used for cultivation; freezing and thawing were repeated three times, and the cells were centrifuged and the supernatant was collected to obtain the amplified oncolytic adenovirus seed solution;

[0011] c. The oncolytic adenovirus seed solution obtained in step b is concentrated by centrifugation and ultrafiltration to obtain the stock solution before column purification;

[0012] d. Separate and purify the stock solution obtained in step c, first use Q Sepharose XL weak anion exchange filler for crude purification, and then use Sourse 30Q or Sourse 15Q strong anion exchange column for fine purification. After two purifications, oncolytic adenovirus is obtained.

[0013] Wherein, in the above-mentioned method for preparing oncolytic adenovirus with improved purification and infection efficiency, the culture in step a adopts DMEM culture medium. Furthermore, the DMEM culture medium is a DMEM culture medium containing 10% fetal bovine serum.

[0014] Wherein, in the above-mentioned method for preparing oncolytic adenovirus with improved purification and infection efficiency, the DMEM culture medium described in step b is a DMEM culture medium containing 2-5% fetal bovine serum.

[0015] Among them, in the above-mentioned oncolytic adenovirus preparation method for improving purification and infection efficiency, the specific operation method of freezing and thawing described in step b is: freezing at -80±2°C and then thawing at 37±1°C.

[0016] Among them, in the above-mentioned oncolytic adenovirus preparation method for improving purification and infection efficiency, the centrifugation described in step c is centrifugation at 4±1°C and 8000±100rpm for 25-35 minutes.

[0017] Among them, in the above-mentioned oncolytic adenovirus preparation method for improving purification and infection efficiency, the specific operation of ultrafiltration concentration described in step c is: collecting the supernatant after centrifugation, filtering and clarifying the virus stock solution through 1.2μm and 0.45μm capsule filters respectively; then concentrating the clarified virus solution by 10 times the volume with a 300KD membrane package; and then filtering and washing the concentrated virus solution with 10 times the volume of filtration and washing buffer.

[0018] Furthermore, the membrane package is a Sartorius Vivaflow 200 tangential flow membrane package Hydrosart membrane with a molecular weight cutoff of 100-500 kDa.

[0019] Furthermore, the filtration and washing buffer solution has a pH of 8.0 and is composed of 20 mM Tris-HCl, 2 mM MgCl 2 , 100-500mM NaCl.

[0020] Among them, in the above-mentioned oncolytic adenovirus preparation method for improving purification and infection efficiency, the specific operation of the crude purification described in step d is: use 5 column volumes of low-salt buffer to balance the separation and purification column, and the equilibrium flow rate is 20 mL / min; after equilibrium, load the sample at 10 mL / min, and after loading, use low-salt buffer to balance to the conductive wire level of salt ions at 15 mL / min; linear gradient elution of the sample, low-salt buffer from 100%-0%, high-salt buffer from 0%-100%, 10 times the column volume to elute the sample, the flow rate is 10 mL / min, and when the A260nm value is greater than A280nm, the peak that appears is collected for the sample; after the sample is collected, the column is regenerated with 2M NaCl buffer for 5 column volumes at a flow rate of 5 mL / min, and the column is stored in 20% alcohol after regeneration.

[0021] Furthermore, the low-salt buffer composition includes: 20 mM Tris-HCl, 2 mM MgCl 2 pH 8.0 ± 0.1.

[0022] Furthermore, the high salt buffer composition includes: 20mM Tris-HCl, 2mM MgCl 2 , 1M NaCl pH 8.0 ± 0.1.

[0023] Among them, in the above-mentioned oncolytic adenovirus preparation method for improving purification and infection efficiency, the operation mode of the fine purification described in step d is the same as the crude purification, using a Sourse 30Q or Sourse 15Q strong anion exchange column.

[0024] Among them, in the above-mentioned oncolytic adenovirus preparation method for improving purification and infection efficiency, the elution mode of the two-step anion exchange chromatography described in step d is linear elution, and the elution buffer is a high salt buffer.

[0025] Among them, in the above-mentioned method for preparing oncolytic adenovirus for improving purification and infection efficiency, the oncolytic adenovirus obtained in step d has a salt ion concentration of 100-1000 mM. The preferred salt ion concentration is 300-800 mM.

[0026] The present invention also provides an oncolytic adenovirus prepared by the above method.

[0027] Wherein, the above-mentioned oncolytic adenovirus contains sodium chloride at a concentration of 100-1000mM.

[0028] Among them, the above-mentioned oncolytic adenovirus is stored at -80°C or -20°C.

[0029] The present invention also provides a use of the oncolytic adenovirus in preparing an anti-tumor drug.

[0030] Wherein, in the above use, the tumor includes all tumors with high expression of telomerase.

[0031] Preferred include breast cancer, melanoma, lung cancer or colon cancer.

[0032] Among them, in the above-mentioned use, the optimal MOI value of the oncolytic adenovirus infection is 10-300.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a method for preparing an oncolytic adenovirus. First, on the basis of the existing three-step method for separating and purifying oncolytic adenovirus, desalting chromatography is reduced, and the existing three-step method for purifying oncolytic adenovirus is reduced to a two-step method by improvement, which greatly reduces the purification time of the virus, reduces the loss of the virus, and enhances the recovery rate of the virus; secondly, the oncolytic adenovirus product obtained by separation and purification by the improved method contains a relatively high concentration of salt ions, the concentration of which is 100-1000mM. Compared with the oncolytic adenovirus obtained by the unimproved method (the salt ion concentration is 0-50mM), the titer, infection activity, particle number, nucleocapsid protein and short-term structural stability of the oncolytic adenovirus are not changed by the salt ion concentration, and high salt ions further enhance the oncolytic adenovirus infection of tumor cells, promote viral replication in tumor cells and enhance the apoptosis of tumor cells; finally, in an in vivo animal experimental model, compared with the unimproved process, the oncolytic adenovirus containing high salt ions obtained by the improved process can better inhibit the growth of tumors, and the tumors of some models have regressed, which greatly improves the survival period of animals. The present invention provides a simple, economical and efficient purification method for large-scale clinical production of oncolytic adenoviruses, and has good application prospects.

[0035] Instruction Manual

[0036] Figure 1 Shown is a statistical curve diagram of different tumor inhibition rates of different salt ions on CT26 cell animal model in Example 1;

[0037] Figure 2 The figure shows the early anti-tumor pharmacodynamics of high salt-promoting oncolytic adenovirus in Example 6; A is the growth curve of the 4T1 breast cancer animal model; B is the survival period of the 4T1 breast cancer animal model; C is the growth curve of the B16F10 melanoma animal model; D is the survival period of the B16F10 melanoma animal model; E is the growth curve of the LLC lung cancer animal model; F is the survival period of the LLC lung cancer animal model;

[0038] Figure 3The figure shows the late anti-tumor pharmacodynamics of high salt-promoted oncolytic adenovirus in Example 7; A is the growth curve of the CT26 early colon cancer animal model; B is the survival period of the CT26 early colon cancer animal model; C is the growth curve of the late colon cancer animal model; D is the survival period of the CT26 late colon cancer animal model;

[0039] Figure 4 The figure shows the blood biochemical test evaluation of the high salt-promoting oncolytic adenovirus treatment of the CT26 animal model in Example 7; A is the statistical analysis of sodium ions; B is the statistical analysis of chloride ions; C is the statistical analysis of potassium ions; D is the statistical analysis of blood creatinine; E is the statistical analysis of aspartate aminotransferase; F is the statistical analysis of alanine aminotransferase; G is the statistical analysis of urea nitrogen;

[0040] Figure 5 The figure shows the safety assessment of high salt ion-promoting oncolytic adenovirus treatment of CT26 animal model in Example 7;

[0041] Figure 6 The figure shows the killing effect of different salt concentrations on different tumor cells in Example 8;

[0042] Figure 7 Shown is a graph showing the infection fluorescence expression of different tumor cells with different oncolytic adenovirus infection multiplicity values ​​in Example 9;

[0043] Figure 8 The figure shows the high salt ion enhancement of oncolytic adenovirus infection efficiency, expression and apoptosis of tumor cells in Example 10; A is a confocal fluorescence imaging of different tumor cells; B is a Western-blot detection of the expression of E1A gene in oncolytic adenovirus of different tumor cells; C is a statistical graph of the expression of eGFP gene detected by flow cytometry of different tumor cells; D is a flow cytometry apoptosis statistical analysis of different tumor cells;

[0044] Fig. 9 The figure shows the virus replication after high salt ion enhanced oncolytic adenovirus infection of tumor cells in Example 10; A is a statistical analysis chart of the infection activity of the virus in the supernatant detected by cell immunohistochemistry; C is a graph showing the virus content in the infected supernatant;

[0045] Fig.10 Shown is the high salt ion enhancement of oncolytic adenovirus fluorescence expression in 3D cell spheroids. DETAILED DESCRIPTION

[0046] The present invention provides a method for preparing an oncolytic adenovirus that improves purification and infection efficiency. On the basis of the existing three-step separation and purification of oncolytic adenovirus, the purification process scheme is improved, the three-step separation and purification is improved to two-step purification, and the desalting chromatography step is removed. Compared with the unimproved method, the oncolytic adenovirus product produced by the improved method for separation and purification contains high salt ions, and the virus is stored at -20°C, 4°C and 37°C respectively; samples are taken at -20°C (0 days, 3 days, 6 days, 9 days, 12 days and 15 days), 4°C (0 days, 3 days, 6 days, 9 days, 12 days and 15 days) and 37°C (0 hours, 2 hours, 6 hours, 12 hours and 24 hours), and the short-term stability of oncolytic adenovirus containing high salt ions is detected by TCID50 method, cell immunohistochemistry method, ultraviolet spectrophotometer method, polyacrylamide gel electrophoresis method, and transmission electron microscopy. The results show that the oncolytic adenovirus prepared by the method of the present invention can be stably produced and stored.

[0047] The present invention provides a method for preparing an oncolytic adenovirus that improves purification and infection efficiency. After improving the separation and purification process of oncolytic adenovirus, the oncolytic adenovirus product obtained contains high salt ions. Compared with oncolytic adenoviruses without high salt ions, oncolytic adenoviruses containing high salt ions can enhance the infection effect of mouse breast cancer cells (4T1), melanoma cells (B16F10), lung cancer cells (LLC) and colon cancer cells (CT26). Confocal microscopy, flow cytometry and Western-blot methods are used to detect the expression of eGFP and E1A genes carried by oncolytic adenoviruses; half tissue culture infection dose method and cell immunohistochemistry are used to detect and analyze the replication of oncolytic adenovirus tumor cells; cell flow cytometry is used to detect the death of tumor cells after infection and replication; similarly, confocal microscopy is used to observe the expression of fluorescent eGFP genes in CT26-RFP and B16F10-RFP cell spheres infected with high-salt oncolytic adenoviruses.

[0048] The present invention provides a method for preparing oncolytic adenovirus with improved purification and infection efficiency. After improving the separation and purification process of oncolytic adenovirus, the oncolytic adenovirus product obtained contains high salt ions. Compared with oncolytic adenovirus without high salt, in early breast cancer animal model (4T1), melanoma animal model (B16F10), lung cancer animal model (LLC) and colon cancer animal model (CT26), oncolytic adenovirus containing high salt ions can well inhibit tumor growth, and mice with some tumor models have tumor regression after treatment, greatly improving survival; tumor volume of early administration: 50-100mm 3 ; Dosage conditions: intratumoral administration once every three days, 1×10 9PFU / mouse, 5 doses in total. In an animal model of advanced colon cancer (CT26), the same oncolytic adenovirus containing high salt ions was able to effectively inhibit tumor growth and improve survival; tumor volume at late administration: 200-300mm 3 ; Dosage conditions: intratumoral administration once every three days, 1×10 9 PFU / mouse, a total of 5 doses. In the animal model of advanced colon cancer, after the treatment, blood was collected from the eye sockets and centrifuged at 1500rpm for 10 minutes, and serum was collected to detect changes in sodium ions, chloride ions, potassium ions, blood creatinine, aspartate aminotransferase, alanine aminotransferase and urea nitrogen in blood biochemistry; then the mouse cervical vertebrae were dislocated to take the heart, liver, spleen, lung and kidney of the mouse, fixed with 4% paraformaldehyde, embedded, sliced, HE stained and imaged for analysis; safety evaluation after treatment with high-salt oncolytic adenovirus.

[0049] The present invention improves the existing two-step anion exchange chromatography and desalting chromatography into two-step anion exchange chromatography, which has simple steps, saves purification time, reduces virus loss, and increases virus recovery. Compared with before the improvement, the oncolytic adenovirus separated and purified after the improvement contains higher salt ions, but the purity, infection activity, virus structure and other stability are consistent. This improvement can not only increase the yield of oncolytic virus, but also exert the pro-inflammatory effect of sodium chloride, thereby further enhancing the anti-tumor effect of oncolytic virus.

[0050] The present invention can better prepare oncolytic adenovirus, reduce the steps of separating and purifying oncolytic adenovirus in large-scale clinical production, enhance the recovery rate of the virus, and thus reduce the cost of purification, making it suitable for process production.

[0051] The present invention simplifies the production of oncolytic adenovirus, is simpler than traditional processes, is easy to operate, saves space, saves cost and time, and the obtained oncolytic adenovirus product has a higher oncolytic effect than traditional processes. Salt ions improve the infection efficiency of oncolytic adenovirus, promote the replication of oncolytic adenovirus in tumor cells, and greatly enhance the oncolytic efficiency of oncolytic adenovirus.

[0052] The present invention is further explained below by examples, but it does not mean that the protection scope of the present invention is limited to the scope of the examples. It can be understood by professionals in the field that the present invention generally and / or specifically describes the materials and experimental methods used in the experiment. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible here.

[0053] The HEK293 cells described in the examples were purchased from the ATCC cell bank, the oncolytic adenovirus was constructed and packaged by our research group, and the remaining experimental consumables and reagents were common commercial products.

[0054] Example 1 Tumor inhibition rate of different concentrations of salt ions on CT26 animal model

[0055] Prepare a series of salt ion buffers such as 250mM, 500mM, 750mM, and 1000mM, and store them in a 4°C refrigerator. Digest the cultured mouse colon cancer cells CT26, count them, and wash them three times with serum-free RPMI 1640 medium. Adjust the cell density to 5×10 6 Cells / mL were inoculated into 6-8 week old Balb / c mice, with an inoculation volume of 100 μL per mouse and a cell count of 5×10 5 When the tumor volume is 50-100mm 3 Buffer solutions with different salt ion concentrations were injected intratumorally with a volume of 100 μL for a total of 5 injections, once every three days. The tumor was measured every three days and the measured tumor data were calculated according to the formula: tumor volume (mm 3 )=0.52×length×width 2 , draw the tumor growth curve and analyze the experimental results. Figure 1 shown.

[0056] The results showed that compared with the control group, different concentrations of salt ions inhibited the growth of the CT26 animal tumor model to varying degrees.

[0057] Example 2 Amplification of oncolytic adenovirus

[0058] The specific steps are as follows:

[0059] a. HEK293 cell recovery: Check the records of preserved cells, take a HEK293 cell from the liquid nitrogen tank, and recover to T75cm 2 Incubate in flask at 37°C, 5% CO 2 Culture in an incubator.

[0060] b. Amplification of oncolytic adenovirus seed fluid: Resuscitate the virus in step a to T75 cm 2 During the subculturing process, a portion of HEK293 cells was subcultured to T182 cm 2 In the square bottle, when the cell density reaches more than 95%, check the storage record of the virus seed liquid, take out the virus seed liquid from the corresponding place of the -80℃ refrigerator, prepare it in 2% DMEM culture medium, add it to the cultured HEK293 cells, and put it in a 37℃, 5% CO2 incubator for culture. Observe under a microscope every day, and when more than 95% of the cells become round and fall off, collect the cells and supernatant, freeze and thaw repeatedly between -80℃ and 37℃ three times to release the virus, centrifuge at 3000rpm for 10 minutes, and collect the supernatant, which is the virus seed liquid for amplifying oncolytic adenovirus in large quantities.

[0061] c. Cell expansion: Resuscitate cells in step a to 75cm 2 During the subculture process, another part of HEK293 cells was taken for further subculture. After the cells were fully grown, they were subcultured to T182 cm 2 , 10-layer cell factory, and continued cultivation in a 5.0L bioreactor. The culture conditions of the bioreactor were: temperature 37°C, pH 7.0-7.2, DO 35-50%, and rotation speed 40-50rpm. Samples were taken every day to detect the morphology of cells on the microcarriers. When 95% of the microcarriers were covered with cells, the next step of the experiment was carried out.

[0062] d. Large-scale amplification of oncolytic adenovirus: The virus seed solution obtained in step b was prepared into 2% DMEM infection virus seed solution, which was added to the HEK293 cells cultured in step c for large-scale amplification. The virus infection multiplicity was approximately 20-30, and the infection volume was 5.0 L. After inoculation, samples were taken every day to observe changes in cell morphology on the cell sphere.

[0063] e. Collecting viruses: When 95% of the HEK293 cells cultured on the microcarriers in step d become round and fall off, stop the culture, collect the cells and culture supernatant, add lysis buffer and lyse at 37°C for 1-2 hours, and collect the supernatant by centrifugation, which is the amplified virus stock solution.

[0064] Example 3 Ultrafiltration Concentration of Oncolytic Adenovirus

[0065] The oncolytic adenovirus stock solution amplified in the above Example 2 was centrifuged at 8000 rpm at 4°C for 30 minutes, the residual precipitate was discarded, the supernatant was collected, and the virus stock solution was filtered through 1.2 μm and 0.45 μm capsule filters to clarify the virus stock solution, and then the clarified virus solution was concentrated by 10 times the volume with a 300KD membrane package, and then the concentrated virus solution was filtered and washed with 10 times the volume of filtration and washing buffer to obtain the stock solution before column purification.

[0066] Example 4 Purification of oncolytic adenovirus

[0067] The virus solution obtained in Example 3 was separated and purified according to the following purification scheme: In the first step of purification (coarse purification), the concentrated adenovirus was purified using Q Sepharose XL weak anion exchange filler to remove more than 90% of the impurities. The specific steps are as follows: First, 5 column volumes of low-salt buffer (20 mM Tris-HCl, 2 mM MgCl 2The method comprises the following steps: a) eluting a column with a pH value of 8.0 and a flow rate of 20 mL / min for balancing the separation and purification column; after balancing, loading the sample at a flow rate of 10 mL / min, and balancing the sample to the level of the conductive wire of the salt ion with a low-salt buffer at a flow rate of 15 mL / min after the loading is completed; eluting the sample with a linear gradient, with a low-salt buffer from 100% to 0%, and a high-salt buffer from 0% to 100%, eluting the sample with 10 times the column volume, and a flow rate of 10 mL / min, and collecting the sample at the peak when the A260nm value is greater than the A280nm; after collecting the sample, regenerating the column with a high-concentration NaCl buffer for 5 column volumes, and a flow rate of 5 mL / min, and storing the column in 20% alcohol after the regeneration is completed. The sample eluted in the first step is diluted in half with a low-salt buffer, and the second step of purification (fine purification) is carried out. Using a Sourse30Q strong anion exchange column, the rest of the method was consistent with the first step of purification to obtain a single peak of eluted oncolytic adenovirus samples; the salt ion concentration and salt ion conductivity were used to calculate the salt ion contained in the oncolytic adenovirus, and the concentration was 423-484mM. Prepare a dilution buffer of the same concentration, dilute the purified oncolytic adenovirus to an appropriate concentration, filter and sterilize, and store 1.0mL / tube in a -80 degree refrigerator for subsequent use.

[0068] Example 5 Stability test of oncolytic adenovirus containing high salt ions

[0069] In the oncolytic adenovirus purification method of the present invention, since desalting chromatography is not performed, the salt ion concentration contained in the oncolytic adenovirus product we obtained is higher than that in the traditional purification method. Therefore, we tested the stability of oncolytic adenovirus under high salt buffer: the stability of oncolytic adenovirus obtained by the improved production process and the traditional production process was evaluated mainly from the following aspects.

[0070] Sample processing of oncolytic adenovirus: The oncolytic adenoviruses obtained by the improved production process and the traditional production process were stored at -20℃, 4℃, and 37℃, respectively. The -20℃ and 4℃ samples were sampled and tested at 3 days, 6 days, 9 days, 12 days, and 15 days, respectively; the 37℃ samples were sampled and tested at 2 hours, 6 hours, 12 hours, and 24 hours, respectively.

[0071] (1) Stability test of infection titer of oncolytic adenovirus (TCID50 method)

[0072] The specific steps are as follows:

[0073] a. Cell culture:

[0074] Take out a tube of HEK 293A cells from the liquid nitrogen tank and resuscitate them to 10 cm 2 The culture dish was placed in 5% CO 2, static culture in a 37°C incubator, and when the cell density reaches 80-90%, cell subculture is performed;

[0075] b. Plate 96-well plate

[0076] The cultured HEK 293A cells were digested with trypsin and counted. The cell concentration was adjusted to 5×10 cells using 2% FBSDMEM medium. 6 / mL, plated in 96-well plates, with 10,000 cells per well, culture volume of 100 μL, placed in 37°C 5% CO 2 Culture statically in an incubator.

[0077] c. Dilution of oncolytic adenovirus

[0078] The oncolytic adenovirus samples treated at different temperatures and for different times were diluted using 2% FBSDMEM culture medium according to the method in Table 1.

[0079] Table 1 Different dilutions of oncolytic adenovirus samples

[0080]

[0081] Note: 1, 10 -1 -10 -13 is the diluted virus gradient;

[0082] 2. 900 μL and 1.17 mL of 2% fetal bovine serum DMEM medium;

[0083] 3. 100 μL and 130 μL are virus dilutions of different gradients;

[0084] d. Add the diluted oncolytic adenovirus dilution solution into the 96-well plate that has adhered to the wall.

[0085] Note: 1. From left to right, 1-10 are 10 repetitions of a virus gradient, and 11-12 are blank controls;

[0086] 2. From top to bottom, select 8 different virus gradients according to the virus titer;

[0087] 3. The total volume of each well is 200 μL, including 100 μL of cells and 100 μL of virus dilution;

[0088] Label (name, date); place in 37°C, 5% CO 2 The cells were cultured in an incubator. On the tenth day, the number of wells in which the oncolytic adenovirus infected 293 cells produced lesions was observed. According to the number of wells with lesions and the formula T = 10 × 10 1+d(S-0.5) TCID50 / mL was used to calculate the titer of oncolytic adenovirus; the changes in infection titer at different temperatures and times were counted and analyzed.

[0089] (2) Activity and stability test of oncolytic adenovirus (cell immunohistochemistry)

[0090] The specific steps are as follows:

[0091] a. Preparation of cell suspension:

[0092] The cultured HEK293 cells were digested with trypsin, counted, and prepared into a cell concentration of 5×10 5 cells / mL of cell suspension, inoculated into a 24-well cell culture plate, 0.5 mL per well;

[0093] b. Sample dilution and inoculation

[0094] The oncolytic adenovirus samples treated at different temperatures and time points were serially diluted 10-fold. -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 The five dilutions of the virus dilutions were inoculated into cells, with each dilution inoculated into two wells, and 50 μL inoculated into each well; after the inoculation, the 24-well cell culture plate was placed in a 37° C., 5% CO 2 incubator for 48 hours.

[0095] c. Cell Fixation

[0096] Aspirate the culture medium in the wells, gently add pre-cooled methanol along the well wall, 500 μL per well, and place at -20°C for 10 minutes. Aspirate the methanol, add 500 μL PBS + 1% BSA to each well for washing, and wash three times.

[0097] d. Primary antibody incubation

[0098] Dilute Mouse-Anti-HexonAntibody 1000 times with PBS + 1% BSA, add 250 μL to each well of a 24-well cell culture plate, and incubate overnight in a 4°C refrigerator. Aspirate Mouse-Anti-HexonAntibody, add 500 μL PBS + 1% BSA to each well for washing, and wash three times.

[0099] e. Secondary antibody incubation

[0100] Dilute Rat-Anti-Mouse Antibody 500 times with PBS + 1% BSA, add to 24-well cell culture plates, 250 μL per well, incubate at 37°C for 1 hour. Aspirate Rat-Anti-Mouse Antibody, add 500 μL PBS + 1% BSA per well, wash three times.

[0101] f. Color rendering

[0102] 10×DAB substrate was diluted 10 times with 1×Stable Peroxidase Buffer, equilibrated to room temperature, added to a 24-well cell culture plate, 250 μL per well, and reacted at room temperature in the dark for 10 minutes.

[0103] g. Stop color development

[0104] Aspirate 1x DAB and add 500 μL PBS to each well.

[0105] h. Cell observation and counting

[0106] Under the 10× eyepiece, adjust the objective lens magnification and the dilution multiple of the sample well so that one field of view contains 5 to 50 positive cells. Count at least 3 fields of view for each plate well and take the average; the average of the positive cells in the two plate wells is the number of infected cells in each field of view at this dilution.

[0107] i. Result counting

[0108] Counting method: Refer to Adeno-X TM Rapid Titer Kit User Manual.

[0109] Virus infection titer (IFU / ml) = (number of infected cells per field of view × number of fields per well) /

[0110] (Volume of virus dilution per well × virus dilution factor)

[0111] (3) Particle number stability test of oncolytic adenovirus

[0112] Take 152 μL of the oncolytic adenovirus at different temperatures and times, add 8 μL of 2% SDS lysis solution and mix well. Centrifuge the test tube containing the above sample briefly in a centrifuge, then place it on the buoyancy plate and incubate it in a 56°C constant temperature water bath for 30 minutes. Take out the sample incubated on the buoyancy plate, wait until it cools to room temperature, centrifuge it instantaneously, and use a UV spectrophotometer to measure the wavelength A 260nm With A 280nm The absorbance value at 10 is calculated according to the formula: Number of virus particles = A260nm × 1.1 × 10 12 × times, calculate the number of virus particles. Count and analyze the changes in the number of particles at different temperatures and times.

[0113] (4) Detection of oncolytic adenovirus nucleocapsid protein

[0114] The specific steps are as follows:

[0115] a. Sample processing:

[0116] Take 40 μL of the oncolytic adenovirus samples containing high salt ions and not containing high salt ions at -20°C, 4°C for 15 days, and 37°C for 24 hours, add 10 μL of 5× loading buffer and mix well, put into 100°C boiling water and heat for 10 minutes; centrifuge at 13000 rpm for 5 minutes to collect the samples.

[0117] b. Polyacrylamide gel electrophoresis:

[0118] Prepare a 12.5% ​​polyacrylamide gel, load a sample of 20 μL, and perform electrophoresis at 80 V for about 20-30 minutes. When the sample enters the separation gel, change the voltage to 120 V. Stop electrophoresis when the bromophenol blue line reaches the bottom. Perform Coomassie brilliant blue staining for 60 minutes, prepare an eluent (Milli-Q: ethanol: glacial acetic acid = 7:2:1) to decolorize until the background is white, and perform imaging analysis.

[0119] (5) Detection of oncolytic adenovirus structure

[0120] Take 20 μL of the above-mentioned oncolytic adenovirus samples containing high salt ions and not containing high salt ions at -20°C, 4°C for 15 days and 37°C for 24 hours, drop them into the copper mesh of the transmission electron microscope, float and stain for 3 minutes, and use absorbent filter paper to absorb the remaining sample. Then add 10-20 μL of phosphotungstic acid to float and stain for 3 minutes, use absorbent filter paper to absorb the remaining phosphotungstic acid, let it stand for 3-5 minutes, install the prepared copper mesh, and perform transmission electron microscope structure detection and analysis.

[0121] In summary, the results show that: when the oncolytic adenovirus samples without high salt ions and with high salt ions were treated at different temperatures and for different times, the infection titer, activity, number of particles, nucleocapsid protein and virus structure were detected. The oncolytic adenovirus samples containing high salt ion buffer were not affected by the concentration of salt ions, and the infection titer, activity, number of particles, nucleocapsid protein and virus structure did not change much. This shows that under high salt ion concentration, the oncolytic adenovirus is still in a stable state and still has infection activity.

[0122] Example 6 Detection of virus loss rate in the oncolytic adenovirus separation and purification step

[0123] Three batches of oncolytic adenovirus samples were produced continuously. According to the oncolytic adenovirus infection titer stability test (TCID50 method) in Example 5, the titers of samples such as concentration, crude purification, fine purification and desalting chromatography during the virus purification process were tested, and the virus loss rate and yield in each purification process were calculated (Table 2).

[0124] Table 2 Recovery rate detection of three consecutive batches of oncolytic adenovirus purification samples

[0125]

[0126]

[0127] The loss of virus and the amount of recovered virus in each step of the purification process were calculated, which proved that the loss of virus was reduced and the recovery rate of virus was increased after the continuous production of three batches of oncolytic adenovirus samples, compared with the purification process without improvement and the improved purification scheme.

[0128] Example 7 High salt promotes the early anti-tumor pharmacodynamics of oncolytic adenovirus

[0129] Cultured mouse lung cancer cells (LLC), breast cancer cells (4T1) and melanoma cells (B16F10) were digested and counted, and washed three times with serum-free RPIM 1640 medium to adjust the cell concentration to 5×10 6 The cells were inoculated into 6-8 week old Balb / c or C57 mice, with an inoculation volume of 100 μL per mouse and an inoculation volume of 5×10 5 When the tumor volume is 50-100mm 3 At the same time, mice were treated with VHSB (salt concentration of 458 mM), OVs (salt concentration of 20 mM), and OVs (VHSB) (salt concentration of 458 mM) by intratumoral injection, respectively. The volume of each mouse was 100 μL, and the virus content was 1.0 × 10 9 PFU, once every three days, a total of 5 times; tumors were measured every three days, and the measured tumor data were calculated according to the formula: tumor volume (mm 3 )=0.52×length×width 2 , draw the tumor growth curve and analyze it. When the tumor volume after treatment is significantly different from that of the control group, stop measuring the tumor volume and observe the survival of mice after treatment, and analyze the survival curve.

[0130] The experimental results are as follows Figure 2 As shown. The results showed that: three types of mouse animal models were established, and statistical analysis after treatment showed that the oncolytic adenovirus containing high salt ions [OVs (VHSB)] had better therapeutic effects than the single-drug group VHSB and OVs, and the difference was significant and statistically significant. After treatment with LLC and B16F10 animal models, some mice tumors regressed. Improved the survival of mice. It further shows that compared with oncolytic adenoviruses without high salt ions, high salt ions promote the therapeutic effect of oncolytic adenoviruses in vivo.

[0131] Example 8 High salt promotes the late anti-tumor pharmacodynamics of oncolytic adenovirus

[0132] Cultured mouse colon cancer cells (CT26) were digested and counted, and washed three times with serum-free RPIM 1640 medium to adjust the cell concentration to 5×10 6 The cells were inoculated into 6-8 week old Balb / c mice, with an inoculation volume of 100 μL per mouse and an inoculation volume of 5×10 5 The tumor volume for early treatment is 50-100mm 3 , the tumor volume for advanced treatment is 200-300mm 3 The mice were treated with VHSB (salt concentration of 458 mM), OVs (salt concentration of 20 mM), and OVs (VHSB) (salt concentration of 458 mM) by intratumoral injection, respectively. The volume of each mouse was 100 μL, and the virus content was 1.0 × 10 9 PFU, once every three days, a total of 5 times; tumors were measured every three days, and the data of tumor measurements were calculated according to the formula: tumor volume (mm 3 )=0.52×length×width 2 , draw the tumor growth curve and analyze and stop the measurement, observe the survival of mice after treatment, and statistically analyze the changes in survival period. After three treatments, 5-6 mice in the late CT26 model were taken from each group, blood was collected from the eye sockets, and serum was obtained by centrifugation at 4°C 2000rpm. The changes in blood biochemistry [sodium ions, chloride ions, potassium ions, blood creatinine, aspartate aminotransferase, alanine aminotransferase and urea nitrogen] were detected, and after cervical dislocation, the heart, liver, spleen, lung and kidney of each mouse were fixed with 4% polyformaldehyde, embedded in paraffin, sliced, and stained with HE. Microscope imaging was used for statistical analysis of the safety of high salt ion buffer, oncolytic adenovirus and oncolytic adenovirus containing high salt ion buffer on mice.

[0133] The experimental results are as follows Figure 3 , 4 and 5.

[0134] The results showed that compared with the CT26 model treated in the early stage, the CT26 model treated in the late stage had the same treatment trend. [OVs (VHSB)] Compared with the single-drug group VHSB and OVs, the oncolytic adenovirus containing high salt ions had better treatment effects than the single-drug group VHSB and OVs. The difference was significant and statistically significant, and the survival period was improved accordingly. Among them, compared with the CT26 model treated in the late stage, the oncolytic adenovirus group containing high salt ions in the CT26 model treated in the early stage had some mouse tumor regression, while in the CT26 model treated in the late stage, there was no tumor regression ( Figure 3 ); Compared with the control group, there was no statistically significant difference in the blood biochemical tests of high salt ion buffer (VHSB), oncolytic adenovirus (OVs) and oncolytic adenovirus containing high salt ion buffer [OVs (VHSB)] ( Figure 4); HE staining analysis of heart, liver, spleen, lung and kidney showed no significant difference between the treatment groups compared with the control group ( Figure 5 ), indicating that local treatment of VHSB, OVs, and OVs (VHSB) is relatively safe and does not cause damage to various organs.

[0135] Example 9 The killing effect of salt ions of different concentrations on tumor cells in vitro

[0136] Cultured mouse colon cancer cells (CT26), lung cancer cells (LLC), breast cancer cells (4T1), and melanoma cells (B16F10) were cultured at 5.0×10 5 Each well was plated in a twelve-well plate and incubated at 37°C with 5% CO 2 After the cells adhered to the wall, the prepared mother solution with a concentration of 1M NaCl buffer was added to the adhered cells according to working concentrations of 50mM, 75mM, 125mM, 150mM, and 175mM and placed in a 37°C, 5% CO 2 Continue to culture in the incubator, observe the cell morphology under a microscope, and take out the cells after 72 hours for the following operations: Cell fixation: discard the culture supernatant, add 1 mL of ice-cold methanol, place in a -20°C refrigerator for 20 minutes, and discard the methanol; Cell washing: slowly wash three times with pure water or PBS buffer; Cell staining: add 0.5 mL of crystal violet to each well and stain for 30 minutes at room temperature; Cell washing: recover the crystal violet and wash three times with pure water or PBS; dry at room temperature and take pictures for analysis;

[0137] The experimental results are as follows Figure 6 shown.

[0138] The results show that different concentrations of salt ions have different killing abilities on different tumor cells. In this study, we selected a suitable concentration that inhibits the growth of tumor cells without causing apoptosis of tumor cells. Therefore, the specific concentrations screened in the present invention are as follows: the salt ion concentration of breast cancer cells (4T1) and melanoma cells (B16F10) is 75mM, the salt ion concentration of colon cancer cells (CT26) is 125mM, and the salt ion concentration of lung cancer cells (LLC) is 150mM.

[0139] Example 10 Infection effect of oncolytic adenovirus with different infection indexes on tumor cells

[0140] Colon cancer cells (CT26), lung cancer cells (LLC), breast cancer cells (4T1) and melanoma cells (B16F10) of mice were cultured at 5.0×10 5 Each well was plated in a twelve-well plate and placed at 37°C with 5% CO 2After the cells adhered to the wall, the adenovirus was diluted with medium containing 2% FBS (DMEM or 1640), and the diluted oncolytic adenovirus was added according to the following multiplicity of infection (MOI: 10, 25, 50, 75, 100, 150, 200, 300). The cells were incubated at 37°C with 5% CO 2 Infect in a small volume in an incubator for 2-6 hours, aspirate the infection solution, replace with medium containing 10% FBS (DMEM or 1640), culture statically for 72 hours, and take photos and analyze under a fluorescence microscope.

[0141] The experimental results are as follows Figure 7 shown.

[0142] The experimental results show that the sensitivity of cells from different mice to oncolytic adenovirus is inconsistent. In the present invention, the oncolytic adenovirus infection multiplicity values ​​selected for the four mouse cells are different; the infection multiplicity value selected for mouse colon cancer cells (CT26) is 200; the infection multiplicity value selected for lung cancer cells (LLC) and breast cancer cells is 10; and the infection multiplicity value selected for melanoma cells (B16F10) is 25.

[0143] Example 11 High salt ions promote the infection efficiency, expression, replication and apoptosis of oncolytic adenovirus in tumor cells

[0144] Colon cancer cells (CT26), lung cancer cells (LLC), breast cancer cells (4T1) and melanoma cells (B16F10) of mice were cultured at a rate of 1.0×10 6 Each well was plated in a six-well plate and placed in a 37°C, 5% CO 2 The cells were cultured statically in an incubator until they adhered to the wall. The salt ion concentration screened in Example 9 and the oncolytic adenovirus infection multiplicity screened in Example 10 were added to the tumor cells, respectively, and placed in an incubator at 37°C and 5% CO 2 The cells were statically cultured in an incubator, and changes in cell fluorescence were observed using a fluorescence microscope for 72 hours. The cell supernatant was collected for detection of viral replication of oncolytic adenovirus in tumor cells. The detection method [1 and 2 in Example 4] was used, and the cell pellet was digested with trypsin and centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded to collect the cell pellet, and the cells were washed three times with PBS (containing 1% FBS). One part was used for Western-blot to detect the expression of oncolytic adenovirus E1A gene in tumor cells, and the other part was stained with cell death Marker (7-AAD). After staining, flow cytometry was used to detect fluorescence expression (eGFP) and cell death (7-AAD).

[0145] The experimental results are as follows Figure 8 and 9shown.

[0146] The experimental results showed that the following methods were used to detect and analyze the gene expression and cell death of oncolytic adenovirus in tumor cells; fluorescence microscopy imaging was used to analyze the expression of oncolytic adenovirus eGFP gene; flow cytometry was used to detect the expression of eGFP gene and cell death in oncolytic adenovirus 7-AAD, and Western-bolt method was used to detect the expression of E1A gene; compared with the single-drug oncolytic adenovirus (OVs-eGFP), the oncolytic adenovirus containing high salt ions [OVs-eGFP (NaCl)] enhanced the expression of E1A gene in tumor cells ( Figure 8 B); Enhanced expression of fluorescent protein eGFP and increased cell death (7-AAD) ( Figure 8 A and 8C, 8D); there is a significant statistical difference analysis; the results show that high salt ions promote the efficiency of oncolytic adenovirus infection of tumor cells and increase the number of dead cells. Using cell immunohistochemistry ( Fig. 9 A) and TCID50 method ( Fig. 9 B) The amount of oncolytic adenovirus in the supernatant of tumor cells infected for 72 hours was detected. Compared with the oncolytic adenovirus (OVs-eGFP) in the single-drug group, the oncolytic adenovirus containing high salt ions [OVs-eGFP (NaCl)] replicated more viruses in tumor cells, with significant statistical differences.

[0147] Example 12 High salt ions promote the expression of oncolytic adenovirus in 3D tumor cell spheroids

[0148] Colon cancer cells (CT26-FRP) and melanoma cells (B16F10-FRP) were revived with FRP red light and placed in a 37°C, 5% CO 2 The cells were cultured statically in an incubator and maintained by adding 2 μg / mL puromycin to the culture medium. The cultured cells were digested with trypsin, counted, and added to a low-adhesion six-well plate. 1×10 4 Place in a 37°C, 5% CO 2 After static culture in an incubator for 10-15 days, when the cells become cell spheres of appropriate size, high salt ion buffer, oncolytic adenovirus, and oncolytic adenovirus containing high salt ions are added to infect the tumor cell spheres, and the expression of the eGFP gene carried by the oncolytic adenovirus in the cell spheres is observed for 72 hours;

[0149] The experimental results are as follows Fig.10 shown.

[0150] The experimental results showed that, compared with the single-drug group oncolytic adenovirus, the oncolytic adenovirus containing high salt ions had enhanced expression of the eGFP gene in colon cancer cell spheres (CT26-FRP) and melanoma cell spheres (B16F10-FRP), and the oncolytic adenovirus containing high salt ions was more likely to enter the cell spheres. This result shows that high salt ions enhance the efficiency of oncolytic adenovirus infection of tumor cell spheres.

Claims

1. Method for preparing oncolytic adenovirus with improved purification and infection efficiency, It is characterized in that The following steps are involved: a. Resuscitate HEK293 cells and incubate at 37±1°C, 5±0.5% CO 2 Cultivated under conditions; b. Prepare new DMEM culture medium, add oncolytic adenovirus seed solution according to the multiplicity of infection of 20-30, and the cells cultured in step a, and incubate at 37±1°C, 5±0.5% CO 2 Conditions were used for cultivation; freezing and thawing were repeated three times, and the cells were centrifuged and the supernatant was collected to obtain the amplified oncolytic adenovirus seed solution; c. The oncolytic adenovirus seed solution obtained in step b is concentrated by centrifugation and ultrafiltration to obtain the stock solution before column purification; d. Separate and purify the stock solution obtained in step c, first use Q Sepharose XL weak anion exchange filler for crude purification, and then use Sourse 30Q or Sourse 15Q strong anion exchange column for fine purification. After two purifications, oncolytic adenovirus is obtained.

2. The method for preparing oncolytic adenovirus with improved purification and infection efficiency according to claim 1, Features: The culture in step a adopts DMEM culture medium; preferably, the DMEM culture medium is DMEM culture medium containing 10% fetal bovine serum.

3. The method for preparing oncolytic adenovirus with improved purification and infection efficiency according to claim 1, Features: The DMEM culture medium described in step b is a DMEM culture medium containing 2-5% fetal bovine serum.

4. The method for preparing oncolytic adenovirus with improved purification and infection efficiency according to claim 1, Features: The specific operation method of freezing and thawing described in step b is: freezing at -80±2°C and then thawing at 37±1°C.

5. The method for preparing oncolytic adenovirus with improved purification and infection efficiency according to claim 1, Features: The centrifugation in step c is carried out at 4±1°C and 8000±100 rpm for 25-35 minutes.

6. The method for preparing oncolytic adenovirus with improved purification and infection efficiency according to claim 1, Features: The specific operation of ultrafiltration concentration described in step c is: collecting the supernatant after centrifugation, filtering and clarifying the virus stock solution through 1.2μm and 0.45μm capsule filters respectively; then concentrating the clarified virus solution by 10 times the volume using a 300KD membrane package; and then filtering and washing the concentrated virus solution with 10 times the volume of filtration and washing buffer.

7. The method for preparing oncolytic adenovirus with improved purification and infection efficiency according to claim 1, Features: The specific operation of the crude purification in step d is: using 5 column volumes of low-salt buffer to equilibrate the separation and purification column, and the equilibrium flow rate is 20 mL / min; after equilibration, loading is performed at 10 mL / min, and after loading, the low-salt buffer is used at 15 mL / min to equilibrate to the conductive wire level of the salt ions; The sample was eluted with a linear gradient, with a low-salt buffer solution from 100% to 0%, and a high-salt buffer solution from 0% to 100%, for 10 times the column volume, at a flow rate of 10 mL / min, and the sample was collected from the peak that appeared when the A260nm value was greater than the A280nm; after the sample was collected, the column was regenerated with a 2M NaCl buffer solution for 5 column volumes at a flow rate of 5 mL / min, and the column was stored in 20% alcohol after the regeneration.

8. The method for preparing oncolytic adenovirus with improved purification and infection efficiency according to claim 1, It is characterized in that Include at least one of the following: The low-salt buffer composition includes: 20mM Tris-HCl, 2mM MgCl 2 pH 8.0 ± 0.1; Or the high salt buffer composition includes: 20mM Tris-HCl, 2mM MgCl 2 , 1 M NaCl pH 8.0 ± 0.1; Or the fine purification described in step d is carried out in the same manner as the crude purification, using a Sourse 30Q or Sourse 15Q strong anion exchange column; Or the elution mode of the two-step anion exchange chromatography described in step d is linear elution, and the elution buffer is a high salt buffer; Or in the oncolytic adenovirus obtained in step d, the salt ion concentration is 100mM-1000mM.

9. An oncolytic adenovirus prepared by the method for preparing an oncolytic adenovirus according to any one of claims 1 to 8.

10. Use of the oncolytic adenovirus according to claim 9 in the preparation of anti-tumor drugs; preferably, the tumor includes breast cancer, melanoma, lung cancer or colon cancer.

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