Preparation and optimization method of purified stock solution of human rotavirus

Abstract

The invention provides a preparation and optimization method of a purified stock solution of a human rotavirus, relates to the technical field of viral vaccines, and provides a human rotavirus production method which comprises the following steps: S1, carrying out primary recovery on Vero cells; s2, carrying out subculture on the Vero cells; s3, infecting the Vero cells with rotavirus; s4, harvesting cultured viruses; s5, clarifying a virus harvest; s6, after clarification, supernate is taken for ultrafiltration; s7, virus purification after ultrafiltration, an optimized production scheme is provided for large-scale production, a phosphate buffer solution containing poloxamer is added during culture virus harvesting, a purification medium in the virus purification process is limited, most of impurities such as host protein and DNA are removed, and the yield is increased. The purification process is applied to preparation of the oral rotavirus attenuated live vaccine for the first time in China, the safety of the vaccine is greatly improved, meanwhile, the production method is suitable for large-scale production, and the production efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of virus vaccines, and in particular to a method for preparing and optimizing a purified stock solution of human rotavirus. Background Art

[0002] Rotavirus (RV) infection has been recognized as the most important cause of severe diarrhea in infants and young children. Severe cases may even cause death in infants and young children. Currently, there is no specific medicine for RV diarrhea, so vaccination is the only feasible way to prevent diarrhea caused by group A rotavirus infection. In January 2013, the WHO clearly recommended in its rotavirus vaccine position paper that all countries should include oral rotavirus vaccines in their national immunization programs as soon as possible, especially countries with high rotavirus enteritis-related mortality rates should give priority to inclusion.

[0003] Currently, RV vaccine is not a vaccine in the immunization program in China. Those approved for marketing include the oral RV live attenuated vaccine (LLR) developed by Lanzhou Institute in 2001, the oral pentavalent reconstituted RV live attenuated vaccine (RV5) launched in 2018, and the oral trivalent reconstituted rotavirus live attenuated vaccine (Vero cell) in 2023. Therefore, there is a huge market gap in my country for rotavirus vaccines, especially independently developed rotavirus vaccines.

[0004] Since oral vaccines need to pass through the digestive system, they have special requirements for the stability of their formula ingredients. Vaccine formulas must ensure the stability of antigen components to ensure that the vaccine has maximum efficacy and antigen activity when used. The genotype of the RV3-BB strain is G3P[6], which was found in the feces of an asymptomatic healthy full-term infant. The RV3-BB strain rotavirus vaccine was developed to prevent severe rotavirus infectious diseases. Clinical research results abroad have confirmed its safety and good immunogenicity. Traditional Vero cell culture and rotavirus culture use trypsin extracted from pig tissue. Trypsin derived from animal tissue is prone to animal-derived virus contamination, which has a serious impact on vaccine safety. For oral attenuated rotavirus vaccines, there are currently no requirements for host cell residues in China.

[0005] The Chinese invention patent application number CN201380054966.7 proposes a modified human rotavirus and its use, a modified Vero-adapted human rotavirus strain and a culture method for producing high-titer virus, a rotavirus vaccine, a vaccination program, and a diagnosis and prognosis assay. This method has low efficiency when used in factory production and cannot meet the needs of large-scale production.

[0006] In order to solve the above problems, it is urgent to develop a method for preparing and optimizing a purified stock solution of human rotavirus to solve the above problems. Summary of the invention

[0007] The present invention discloses a method for preparing and optimizing a purified stock solution of human rotavirus, which ensures the virus recovery rate, removes residual DNA and protein of host cells, replaces the virus culture solution with a phosphate buffer solution with a clear formula, makes the attenuated live rotavirus vaccine meet the requirements of the European Pharmacopoeia, and improves the safety of the vaccine.

[0008] To achieve the above objectives, the present invention is implemented by the following technical scheme: a preparation method of a purified human rotavirus stock solution and a large-scale production and optimization method, comprising the following steps:

[0009] S1, primary recovery of Vero cells;

[0010] S2, Vero cell subculture;

[0011] S3, rotavirus infection of Vero cells;

[0012] S4, culture virus harvest;

[0013] S5, clarification of viral harvest;

[0014] S6, after clarification, take the supernatant and ultrafilter;

[0015] S7, virus purification after ultrafiltration;

[0016] In S2, recombinant trypsin with an added concentration of 0.25% EDTA was used to digest the Vero cells, and the cell fluid after digestion was passaged in proportion.

[0017] Among them, the large-scale production optimization methods include the optimization of virus large-scale culture process and the optimization of virus stock solution preparation process.

[0018] Preferably, the virus scale culture process is optimized, including virus activation, virus adsorption and virus culture methods, the virus in S3 is RV3-BB virus, the virus scale culture container is a ten-layer cell factory, and the virus infection volume is 1-10 ml / ten-layer cell factory.

[0019] Preferably, the virus activation scheme is to use 100U / mL-200U / mL of 0.25% EDTA recombinant pancreatic enzyme for virus activation, the activation temperature is 37±1°C, and the activation time is 30-90min.

[0020] Preferably, the virus adsorption method is to dilute the activated virus and supplement the DMEM culture medium with the same concentration as the diluent until the total volume of the solution is 40 ml / layer, and then adsorb the virus for 2 hours.

[0021] Preferably, the virus culture method is to supplement DMEM to a total volume of 200 ml / layer after virus adsorption, and perform virus culture for 2-5 days.

[0022] Preferably, the optimization of the virus stock solution preparation process includes the optimization of the virus harvest preparation method and the virus purification process.

[0023] Preferably, the method for preparing the virus harvest in S4-S6 is to harvest the virus in a PC bottle, freeze it in an environment with a temperature of -20°C or below, freeze and thaw it twice, centrifuge it at 4000g for 20 minutes, remove cell debris, take the supernatant as the virus harvest, and store it in a sterile environment at 2-8°C.

[0024] Preferably, poloxamer-188 is added to the virus harvest, and the concentration of poloxamer-188 is 0.01%-0.1% g / v.

[0025] Preferably, the ultrafiltration membrane package used for virus concentration in S6 is 300 kD in size and has an area of ​​0.1 square meters, and the virus harvest is concentrated 5X, 10X and 10X by replacing 5CV through the ultrafiltration membrane package.

[0026] Preferably, the purification medium used for virus purification in S7 can be one of Max Coll 700, 4FF, Smac Core700 and Diamond Layer 700.

[0027] Preferably, the virus harvest is concentrated 5X by ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Diamond Layer 700 to recover a virus yield of 79%-100%;

[0028] The virus harvest is concentrated 10X through an ultrafiltration membrane package, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Diamond Layer 700 to obtain a virus yield of 45%-100%.

[0029] Beneficial Effects

[0030] The present invention provides a method for preparing and optimizing a purified stock solution of human rotavirus, which has the following beneficial effects compared with the existing technology:

[0031] The rotavirus vaccine developed by the present invention is the first monovalent human rotavirus vaccine in China, which realizes the adaptation and large-scale culture of human rotavirus on Vero cells and realizes the replacement of animal-free pancreatic leukase in the cell and virus culture process.

[0032] The present invention uses recombinant pancreatic enzymes instead of traditional trypsin in the cell culture and virus culture stages, thus avoiding porcine virus contamination from the root, saving detection costs and reducing production risks. Virus culture fluid contains complex metabolites. If the virus culture fluid is directly used for vaccine preparation, the vaccine will include a large number of unknown metabolites.

[0033] The present invention purifies the virus culture fluid and replaces it with a phosphate buffer with a clear formula, thereby avoiding the contamination of porcine viruses from the source. In order to control the contamination of exogenous viruses, the finished product needs to be tested and can only be released after passing the test. The virus harvest is purified according to the European Pharmacopoeia standards to remove most of the impurities such as host proteins and DNA. This is the first time in China that the purification process is applied to the preparation of an oral attenuated live rotavirus vaccine, greatly improving the safety of the vaccine.

[0034] The virus harvested material of the present invention is concentrated 5X by ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer can be purified by Diamond Layer 700 to recover the virus yield of 79%-100%, which has high virus recovery efficiency and improves production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0036] Figure 1 This is a flow chart of rotavirus production of the present invention;

[0037] Figure 2 The fluorescence titer detection bar graph of the purification process of rotavirus (RV3-BB) of the present invention by adding different concentrations of poloxamer;

[0038] Figure 3 A line graph showing the fluorescence titer detection of the purification process for the rotavirus (RV3-BB) of the present invention by adding different concentrations of poloxamer; DETAILED DESCRIPTION

[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail in conjunction with the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The technical solutions of the present invention are described in detail below in conjunction with the embodiments and the accompanying drawings, but the scope of protection is not limited thereto.

[0040] See also Figure 1-3 The present invention provides a technical solution: a method for preparing a purified stock solution of human rotavirus and a method for optimizing large-scale production, comprising the following steps:

[0041] S1, primary recovery of Vero cells;

[0042] S2, Vero cell subculture;

[0043] S3, rotavirus infection of Vero cells;

[0044] S4, culture virus harvest;

[0045] S5, clarification of viral harvest;

[0046] S6, after clarification, take the supernatant and ultrafilter;

[0047] S7, virus purification after ultrafiltration;

[0048] Among them, in S2, recombinant pancreatic enzyme with an added concentration of 0.25% EDTA was used to digest Vero cells to avoid porcine virus contamination from the source, and the digested cell fluid was passaged in proportion.

[0049] Among them, the large-scale production optimization methods include the optimization of virus large-scale culture process and the optimization of virus stock solution preparation process.

[0050] In some embodiments, the virus scale culture process is optimized, including virus activation, virus adsorption and virus culture methods, the virus in S3 is RV3-BB virus, the virus scale culture container is a ten-layer cell factory, and the virus infection volume is 1-10 ml / ten-layer cell factory.

[0051] In some embodiments, the virus activation scheme is to use 100U / mL-200U / mL of 0.25% EDTA recombinant trypsin for virus activation, the activation temperature is 37±1°C, and the activation time is 30-90min.

[0052] In some embodiments, the virus adsorption method is to dilute the activated virus and supplement the DMEM culture medium with the same concentration of the dilution solution to a total volume of 40 ml / layer for adsorption for 2 hours.

[0053] In some embodiments, the virus culture method is to supplement DMEM to a total volume of 200 ml / layer after virus adsorption, and perform virus culture for 2-5 days.

[0054] In some embodiments, the optimization of the virus stock solution preparation process includes the optimization of the virus harvest preparation method and the virus purification process. The virus harvest preparation method in S4-S6 is to harvest the virus in a PC bottle, freeze it in an environment with a temperature of -20°C or below, freeze and thaw it twice, centrifuge it at 4000g for 20 minutes, remove cell debris, take the supernatant as the virus harvest, and store it in a sterile environment at 2-8°C.

[0055] In some embodiments, poloxamer-188 is added to the viral harvest and the poloxamer-188 concentration is 0.01%-0.1% (g / v).

[0056] In some embodiments, the ultrafiltration membrane package used for virus concentration in S6 is 300 kD in size and has an area of ​​0.1 square meters. The virus harvest is concentrated 5X, 10X and 10X replaced by 5CV through the ultrafiltration membrane package.

[0057] In some embodiments, the purification medium used for virus purification in S7 can be one of Max Coll 700, 4FF, SmacCore 700 and Diamond Layer 700.

[0058] Example 1

[0059] The virus concentration process is to concentrate the above virus harvest by 5X through an ultrafiltration membrane package, and the fluorescence titer detection of the phosphate virus concentrate containing 0.01%-0.1% poloxamer is 0.4-4 times better than the fluorescence titer detection of the virus concentrate containing only phosphate.

[0060] Example 2

[0061] The virus concentration process is to concentrate the above virus harvest by 10X through an ultrafiltration membrane package, and the fluorescence titer detection of the phosphate virus concentrate containing 0.01%-0.1% poloxamer is 0.1 times-3 times better than the fluorescence titer detection of the virus concentrate containing only phosphate.

[0062] Example 3

[0063] The virus concentration process is to concentrate the above virus harvest through an ultrafiltration membrane package 10X to replace 5CV, and the fluorescence titer detection of the phosphate virus concentrate containing 0.01%-0.1% poloxamer is 0.03 times-2.3 times better than the fluorescence titer detection of the virus concentrate containing only phosphate.

[0064] The initial number of virus strains was 756,250. After adding different amounts of poloxamer-188, different concentration ratios were used to obtain specific experimental data, see Table 1 below.

[0065] Table 1: Data table of the number of virus strains harvested by adjusting the corresponding experimental ratio under the same virus base number

[0066]

[0067] Comparative Examples 1 to 3 show that when the virus harvest is concentrated 5X by ultrafiltration membrane bag, the fluorescence titer detection of the phosphate virus concentrate containing 0.1% poloxamer is 4 times better than the fluorescence titer detection of the virus concentrate containing only phosphate, indicating a good virus harvest.

[0068] Example 4

[0069] In step S7, the medium selected for purification chromatography is Max Coll 700, and the virus purification process is to concentrate the virus harvest by 5X through an ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Max Coll 700 to prepare a recoverable virus yield of 37%-49%.

[0070] Example 5

[0071] In step S7, the medium selected for purification chromatography is Max Coll 700, and the virus purification process is to concentrate the above virus harvest by 10X through an ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer can be purified by MaxColl 700 to recover a virus yield of 25%-29%.

[0072] Example 6

[0073] In step S7, the medium selected for purification chromatography is Max Coll 700, and the virus purification process method is to concentrate the above-mentioned virus harvest through an ultrafiltration membrane bag 10X replacement 5CV, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Max Coll 700. The virus yield can be recovered at 6%-17%.

[0074] When the medium used for purification chromatography is Max Coll 700, the virus harvest percentage when different concentrations of Poloxamer-188 are used for concentration at different ratios is shown in Table 2 below:

[0075] Table 2: Percentage of virus harvest when the purification chromatography medium is Max Coll 700

[0076]

[0077]

[0078] From the above table, we know that when the medium selected for purification chromatography is Max Coll 700, the concentration is 0.1% poloxamer and the concentration is 5X, the virus harvest extraction rate is 49%, which is the best result of the experiment.

[0079] Example 7

[0080] In step S7, the medium selected for purification chromatography is 4FF, and the virus purification process is to concentrate the above-mentioned virus harvest by 5X through an ultrafiltration membrane bag. The phosphate virus concentrate containing 0.01%-0.1% poloxamer can be purified by 4FF to recover a virus yield of 20%-40%.

[0081] Example 8

[0082] In step S7, the medium selected for purification chromatography is 4FF, and the virus purification process is to concentrate the above-mentioned virus harvest by 10X through an ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer can be purified by 4FF to obtain a recoverable virus yield of 10%-20%.

[0083] Example 9

[0084] In step S7, the medium selected for purification chromatography is 4FF, and the virus purification process is to concentrate the above-mentioned virus harvest through an ultrafiltration membrane bag 10X replacement 5CV, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by 4FF and the virus yield can be recovered at 0.3%-1%.

[0085] When 4FF medium is used for purification chromatography, the virus harvest percentage when different concentrations of poloxamer-188 are used for concentration at different ratios is shown in Table 3 below.

[0086] Table 3: Percentage of virus harvest when 4FF medium is used for purification chromatography

[0087]

[0088] From the above table, we know that when the medium selected for purification chromatography is 4FF, the concentration is 0.01% poloxamer and the concentration is 5X, the virus harvest extraction rate is 40%, which is the best experimental result.

[0089] Example 10

[0090] In step S7, the medium selected for purification chromatography is Smac Core 700, and the virus purification process is to concentrate the above virus harvest by 5X through an ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by SmacCore 700 to recover a virus yield of 62%-100%.

[0091] Embodiment 11

[0092] In step S7, the medium selected for purification chromatography is Smac Core 700, and the virus purification process is to concentrate the above virus harvest by 10X through an ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by SmacCore 700 to recover a virus yield of 33%-61%.

[0093] Example 12

[0094] In step S7, the medium selected for purification chromatography is Smac Core 700, and the virus purification process method is to concentrate the above virus harvest through an ultrafiltration membrane bag 10X replacement 5CV, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Smac Core 700 to recover a virus yield of 25%-30%.

[0095] When the medium used for purification chromatography is Smac Core 700, the virus harvest percentage when different concentrations of poloxamer-188 are used for concentration at different ratios is shown in Table 4 below.

[0096] Table 4: Percentage of virus harvest when the medium used for purification chromatography is Smac Core 700

[0097]

[0098]

[0099] The above table shows that when the medium selected for purification chromatography is Smac Core 700, the concentration is 0.1% poloxamer and the concentration is 5X, the virus harvesting and extraction rate is 100%, which is the best result of the experiment.

[0100] Embodiment 13

[0101] In step S7, the medium selected for purification chromatography is Diamond Layer 700, and the virus purification process method is to concentrate the above-mentioned virus harvest by 5X through an ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer can be purified by Diamond Layer 700 to prepare a recoverable virus yield of 79%-100%.

[0102] Embodiment 14

[0103] In step S7, the medium selected for purification chromatography is Diamond Layer 700, and the virus purification process method is to concentrate the above-mentioned virus harvest by 10X through an ultrafiltration membrane bag, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer can be purified by Diamond Layer 700 to recover a virus yield of 45%-100%.

[0104] Embodiment 15

[0105] In step S7, the medium selected for purification chromatography is Diamond Layer 700, and the virus purification process method is to concentrate the above-mentioned virus harvest through an ultrafiltration membrane package 10X replacement 5CV, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Diamond Layer700. The virus yield can be recovered at 22%-44%.

[0106] When Diamond Layer 700 is used as the medium for purification chromatography, the virus harvest percentages when different concentrations of Poloxamer-188 are used for concentration at different ratios are shown in Table 5 below.

[0107] Table 5: Percentage of virus harvest when Diamond Layer 700 is used as the medium for purification chromatography

[0108]

[0109] From the above table, we know that when Diamond Layer 700 is selected as the medium for purification chromatography, the concentration is 0.1% poloxamer and the concentration is 5X, and the concentration is 0.1% poloxamer and the concentration is 10X, the virus harvest extraction rate is 100%, which is the best experimental result.

[0110] Among them, the histogram of fluorescence titer detection of rotavirus (RV3-BB) purification process with different concentrations of poloxamer added under different concentration conditions is shown in Figure 2 It was found that rotavirus (RV3-BB) achieved better results when 0.1% poloxamer phosphate buffer was added and concentrated 10X.

[0111] Among them, the line graph of the fluorescence titer detection of the purification process when rotavirus (RV3-BB) was added with different concentrations of poloxamer and the purification chromatography medium was selected under different concentration conditions is shown in Figure 3It was found that when rotavirus (RV3-BB) was added with 0.1% poloxamer phosphate buffer and concentrated by 5X, both Smac Core 700 and Diamond Layer 700 could achieve a 100% virus harvest effect. When 0.1% poloxamer phosphate buffer was added and concentrated by 10X, Diamond Layer700 could achieve a 100% virus harvest effect.

[0112] In summary, the scheme of the present invention uses recombinant trypsin instead of traditional trypsin to avoid porcine virus contamination from the source, rotavirus (RV3-BB) is concentrated 10X by adding 0.1% poloxamer phosphate buffer, and the purification medium is Diamond Layer 700 for virus harvesting, which can achieve better production results.

[0113] The above content is a further detailed description of the present invention in combination with a specific preferred embodiment. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the present invention, which should be regarded as belonging to the present invention and the scope of patent protection determined by the submitted claims.

Claims

1. A method for preparing a purified stock solution of human rotavirus and a method for large-scale production and optimization, characterized in that: The following steps are involved: S1, primary recovery of Vero cells; S2, Vero cell subculture; S3, rotavirus infection of Vero cells; S4, culture virus harvest; S5, clarification of viral harvest; S6, after clarification, take the supernatant and ultrafilter; S7, virus purification after ultrafiltration; In S2, recombinant trypsin with an added concentration of 0.25% EDTA was used to digest the Vero cells, and the cell fluid after digestion was passaged in proportion. Among them, the large-scale production optimization methods include the optimization of virus large-scale culture process and the optimization of virus stock solution preparation process.

2. The method for optimizing the large-scale production of a purified human rotavirus stock solution according to claim 1, characterized in that: The virus scale culture process is optimized, including virus activation, virus adsorption and virus culture methods. The virus in S3 is RV3-BB virus, the virus scale culture container is a ten-layer cell factory, and the virus infection volume is 1-10 ml / ten-layer cell factory.

3. The method for optimizing the large-scale production of a purified human rotavirus stock solution according to claim 2, characterized in that: The virus activation scheme is to use 100U / mL-200U / mL of 0.25% EDTA recombinant pancreatic enzyme to activate the virus, the activation temperature is 37±1°C, and the activation time is 30-90min.

4. The method for optimizing the large-scale production of a purified human rotavirus stock solution according to claim 3, characterized in that: The virus adsorption method is to dilute the activated virus, and add DMEM culture medium with the same concentration as the diluent to a total volume of 40 ml / layer, and then adsorb for 2 hours.

5. The method for optimizing the large-scale production of a purified human rotavirus stock solution according to claim 4, characterized in that: The virus culture method is to supplement DMEM to a total solution volume of 200 ml / layer after virus adsorption, and perform virus culture for 2-5 days.

6. The method for optimizing the large-scale production of a purified human rotavirus stock solution according to claim 1, characterized in that: The optimization of the virus stock solution preparation process includes the optimization of the virus harvest preparation method and the virus purification process.

7. The method for optimizing the large-scale production of purified human rotavirus stock solution according to claim 6, characterized in that: The method for preparing the virus harvest in S4-S6 is to harvest the virus in a PC bottle, freeze it in an environment with a temperature of -20°C or below, freeze and thaw it twice, centrifuge it at 4000g for 20 minutes, remove cell debris, take the supernatant as the virus harvest, and store it in a sterile environment at 2-8°C.

8. The method for optimizing the large-scale production of purified human rotavirus stock solution according to claim 7, characterized in that: Poloxamer-188 was added to the virus harvest, and the concentration of Poloxamer-188 was 0.01%-0.1% g / v.

9. The method for optimizing the large-scale production of purified human rotavirus stock solution according to claim 8, characterized in that: The ultrafiltration membrane package used for virus concentration in the S6 is 300 kD in size and has an area of ​​0.1 square meters. The virus harvest is concentrated 5X, 10X and 10X by replacing 5CV through the ultrafiltration membrane package.

10. The method for optimizing the large-scale production of purified human rotavirus stock solution according to claim 1, characterized in that: The purification medium used for virus purification in S7 can be one of Max Col 700, 4FF, Smac Core 700 and DiamondLayer 700; The virus harvest is concentrated 5X by ultrafiltration membrane pack, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Diamond Layer 700 to obtain a recoverable virus yield of 79%-100%; The virus harvest is concentrated 10X through an ultrafiltration membrane package, and the phosphate virus concentrate containing 0.01%-0.1% poloxamer is purified by Diamond Layer 700 to obtain a virus yield of 45%-100%.

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