Large-scale preparation method of parotitis virus with low serum residue

Abstract

The invention relates to the technical field of biology, in particular to a large-scale preparation method of mumps viruses with low serum residues. The method specifically comprises the following steps: digesting resuscitated human diploid cells by using protease, and sequentially culturing in a cell culture solution 01, a cell culture solution 02 and a cell culture solution 03; placing the mumps virus seed in the human diploid cell obtained by culturing, and then placing the human diploid cell in a virus maintenance solution for culturing, so as to obtain a collected supernatant, namely a virus harvesting solution. By optimizing the production process, the pollution risk in the production process is remarkably reduced, the working time is greatly shortened, the overall working efficiency is effectively improved, resource utilization is more efficient, meanwhile, bovine serum residues of the genotype F mumps attenuated live vaccine are reduced, and the production cost is reduced. The potential risk of the bovine serum is avoided; meanwhile, the titer of the cultured virus is not influenced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for large-scale preparation of mumps virus with low serum residue. Background Art

[0002] Epidemic parotitis is an acute respiratory infectious disease caused by mumps virus infection, which is transmitted through the air and mainly occurs in children and adolescents. The incidence of epidemic parotitis is high and the infectivity is strong. It has ranked second in the reported cases of Class C infectious diseases for many consecutive years, and its complications can infect the central nervous system and lead to death. Since the 1960s, with the widespread application of mumps attenuated live vaccine, mumps infection has been effectively controlled.

[0003] Mumps virus is divided into 12 genotypes, and its distribution has obvious regional characteristics. The main epidemic strains in Europe are A, C, D, G, H, the main epidemic strains in the Americas are C, D, G, H, J, K, and the main epidemic strains in Asia are B, F, I, L.

[0004] In recent years, with the increasing demand for various biological products such as vaccines and therapeutic protein drugs in the market and the improvement of product quality, the technology of large-scale cell culture has been continuously improved. Human diploid cells are the main cell matrix for the production of viral vaccines. Since the establishment of the human diploid cell line WI-38 in 1961 and its application in the preparation of rabies vaccine, it has developed rapidly due to its good immunogenicity and safety, and has been widely used in the industrial production of vaccines. Subsequently, many human embryonic diploid cell lines have been established, making great contributions to public health.

[0005] The KMB-17 cell line was established from the embryonic lung tissue of a 4-month-old embryo in 1981. This cell fully conforms to the limited life characteristics of diploid cells. Compared with WI-38 and MRC-5 cells, the probabilities of polyploid and structural abnormalities of the KMB-17 cell line are relatively low, and this cell line has no exogenous factors, no potential carcinogenicity, and is sensitive to a variety of viruses.

[0006] During the early cell culture process of KMB-17 cells, newborn bovine serum can provide necessary nutrients for the cells, such as growth factors, hormones, minerals, etc., to maintain cell growth and reproduction. After the vaccine production is completed, a small amount of bovine serum components will remain in the vaccine product. Residual bovine serum may cause allergic reactions: Bovine serum contains various protein components. For some people with allergic constitutions, these residual bovine serum proteins may trigger allergic reactions. In mild cases, symptoms such as skin itching, erythema, and urticaria may occur, and in severe cases, life-threatening conditions such as anaphylactic shock may result. There is also a risk of introducing other pathogens: If the source animals of bovine serum are infected with certain viruses, bacteria, or other pathogens (such as the mad cow disease virus, although there are currently strict detection measures for bovine serum to prevent such situations, the risk still exists potentially), these pathogens may enter the vaccine through the residual bovine serum and then cause diseases in the vaccinated population. In order to ensure the safety and effectiveness of the vaccine, reduce the residual amount of bovine serum, and make it meet safety standards, the previous production process had the risks of time-consuming, reducing production efficiency, and being easily contaminated. Therefore, how to further improve production efficiency while reducing the risk of contamination and further reducing the use of bovine serum in the culture medium is an urgent problem for those skilled in the art to solve. Summary of the Invention

[0007] The object of the present invention is to provide a method for large-scale preparation of mumps virus with low serum residue.

[0008] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for large-scale preparation of mumps virus with low serum residue, including the following steps:

[0010] (1) Digest the resuscitated human diploid cells with protease, place them in cell culture medium 01, and perform subculture;

[0011] (2) Place the human diploid cells obtained by subculture in step (1) in cell culture medium 02, and perform subculture;

[0012] (3) Place the human diploid cells obtained by subculture in step (2) in cell culture medium 03 for culture;

[0013] (4) Place the mumps virus seed in the human diploid cells obtained by culture in step (3), then place them in virus maintenance fluid for culture, and the harvested supernatant is the virus harvest fluid;

[0014] The cell culture medium 01 is based on MEM solution and includes the following components: glutamine 1.8 - 2.2% (V / V), newborn bovine serum 9 - 11% (V / V), sodium bicarbonate 2 - 4% (V / V);

[0015] The cell culture medium 02 is based on MEM solution and includes the following components: glutamine 1.8 - 2.2% (V / V), newborn bovine serum 3 - 5% (V / V), sodium bicarbonate 2 - 4% (V / V).

[0016] The cell culture medium 03 is based on MEM solution and consists of the following components: glutamine 1.8 - 2.2% (V / V), sodium bicarbonate 2 - 4% (V / V).

[0017] The virus maintenance fluid is based on MEM solution and consists of the following components: glutamine 1.8 - 2.2% (V / V), sodium bicarbonate 4 - 6% (V / V).

[0018] Preferably, the human diploid cell is KMB17 cell; the mumps virus seed is mumps virus SP - A strain of F genotype; the mass percentage concentration of glutamine is 2.5 - 3.5%; the mass percentage concentration of sodium bicarbonate is 6 - 7%.

[0019] Preferably, the passage number of the human diploid cells in step (1) is the 16th - 28th passage.

[0020] Preferably, the human diploid cells passed on in step (2) are at the 32nd passage.

[0021] Preferably, the culture time in step (3) is 3 - 5 d; the temperature is 36.5 - 37.5 °C.

[0022] Preferably, the culture time in step (4) is 7 - 9 d; the temperature is 36.5 - 37.5 °C; in step (4), the mumps virus seed is inoculated according to the MOI value of 0.02 - 0.2.

[0023] Preferably, when placing the cells in the cell culture medium 02 in step (2), the cells need to be washed with a buffer first; when placing the cells in the cell culture medium 03 in step (3), the cells need to be washed with a buffer first.

[0024] Preferably, the buffer is prepared from the following components by mass concentration: NaCl 7 - 9 g / L, KCl 0.1 - 0.3 g / L, Na2HPO4·12H2O 2 - 3 g / L, and KH2PO4 0.1 - 0.3 g / L, with a pH of 7.3 - 7.5.

[0025] The present invention also provides a virus harvest fluid prepared by the preparation method described above.

[0026] The present invention also provides the application of the virus harvest fluid in the preparation of mumps attenuated live vaccine.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] By optimizing the production process, the present invention significantly reduces the pollution risk during production, greatly shortens the working hours, and effectively improves the overall working efficiency. While making resource utilization more efficient, it reduces the residual bovine serum in the live attenuated mumps vaccine of F genotype, avoids the potential risks of bovine serum, and has no impact on the virus titer of the cultured virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0030] Figure 1 It is a schematic diagram of cell passage. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a method for large-scale preparation of mumps virus with low serum residue, which includes the following steps:

[0032] (1) Digest the resuscitated human diploid cells with protease, place them in cell culture medium 01, and perform passage;

[0033] (2) Place the human diploid cells obtained by passage in step (1) in cell culture medium 02, and perform passage;

[0034] (3) Place the human diploid cells obtained by passage in step (2) in cell culture medium 03 for culture;

[0035] (4) Place the mumps virus seed in the human diploid cells obtained by culture in step (3), then place it in virus maintenance medium for culture, and the harvested supernatant is the virus harvest fluid;

[0036] In the present invention, the cell culture medium 01 is based on MEM solution and includes the following components: glutamine 1.8 - 2.2% (V / V), newborn bovine serum 9 - 11% (V / V), sodium bicarbonate 2 - 4% (V / V); preferably glutamine 1.9 - 2.1% (V / V), newborn bovine serum 10% (V / V), sodium bicarbonate 3% (V / V); further preferably glutamine 2% (V / V), newborn bovine serum 10% (V / V), sodium bicarbonate 3% (V / V).

[0037] In the present invention, the cell culture medium 02 is based on MEM solution and comprises the following components: glutamine 1.8 - 2.2% (V / V), newborn bovine serum 3 - 5% (V / V), sodium bicarbonate 2 - 4% (V / V); preferably, glutamine 1.9 - 2.1% (V / V), newborn bovine serum 4% (V / V), sodium bicarbonate 3% (V / V); more preferably, glutamine 2% (V / V), newborn bovine serum 4% (V / V), sodium bicarbonate 3% (V / V).

[0038] In the present invention, the cell culture medium 03 is based on MEM solution and consists of the following components: glutamine 1.8 - 2.2% (V / V), sodium bicarbonate 2 - 4% (V / V); preferably, glutamine 1.9 - 2.1% (V / V), sodium bicarbonate 3% (V / V); more preferably, glutamine 2% (V / V), sodium bicarbonate 3% (V / V).

[0039] In the present invention, the virus maintenance medium is based on MEM solution and consists of the following components: glutamine 1.8 - 2.2% (V / V), sodium bicarbonate 4 - 6% (V / V); preferably, glutamine 1.9 - 2.1% (V / V), sodium bicarbonate 5% (V / V); more preferably, glutamine 2% (V / V), sodium bicarbonate 5% (V / V).

[0040] In the present invention, the human diploid cell is KMB17 cell; the mumps virus seed strain is mumps virus SP - A strain of F genotype;

[0041] In the present invention, the mass percentage concentration of glutamine is 2.5 - 3.5%; preferably 3%.

[0042] In the present invention, the mass percentage concentration of sodium bicarbonate is 6 - 7%; preferably 6.5%.

[0043] In the present invention, the passage number of the human diploid cells in step (1) is the 16th - 28th passage; preferably the 16th - 26th passage; more preferably the 16th - 24th passage; most preferably the 16th passage.

[0044] In the present invention, the human diploid cells passed in step (2) are at the 32nd passage.

[0045] In the present invention, the culture time in step (3) is 3 - 5 d; the temperature is 36.5 - 37.5 °C; preferably the culture time is 4 d; the temperature is 37 °C.

[0046] In the present invention, the culture time in step (4) is 7 - 9 d; the temperature is 36.5 - 37.5 °C; preferably the culture time is 8 d; the temperature is 37 °C.

[0047] In the present invention, in step (4), the mumps virus seed is inoculated according to an MOI value of 0.02 - 0.2; preferably 0.019 - 0.021; more preferably 0.02.

[0048] In the present invention, in step (2), when the cells are placed in cell culture medium 02, the cells need to be washed with a buffer first; in step (3), when the cells are placed in cell culture medium 03, the cells need to be washed with a buffer first.

[0049] In the present invention, the buffer is prepared from the following components by mass concentration: 7 - 9 g / L of NaCl, 0.1 - 0.3 g / L of KCl, 2 - 3 g / L of Na2HPO4·12H2O, and 0.1 - 0.3 g / L of KH2PO4, with a pH of 7.3 - 7.5; preferably 8 g / L of NaCl, 0.2 g / L of KCl, 2.5 g / L of Na2HPO4·12H2O, and 0.2 g / L of KH2PO4, with a pH of 7.4.

[0050] The present invention also provides a virus harvest fluid prepared by the preparation method described above.

[0051] The present invention also provides the application of the virus harvest fluid in the preparation of a live attenuated mumps vaccine.

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

[0053] MEM medium (Gibco, USA); newborn bovine serum (Lanzhou Minhai Bioengineering Co., Ltd.); 3% glutamine (Sigma, USA); 6.6% NaHCO3 (Zigong Honghe Pharmaceutical Co., Ltd.).

[0054] Example 1

[0055] 1. Production cells

[0056] The production cells are human embryonic lung diploid cells KMB17 (human diploid cell), which are preserved by the Institute of Medical Biology, Chinese Academy of Medical Sciences. The cell passage of the original cell bank (PCB) of the KMB17 strain is the 16th passage.

[0057] 2. Cell preparation

[0058] After reviving several ampoules of cells from the working cell bank, inoculate them into T225 cell culture flasks, and use cell culture medium as the cell growth medium for subculture and amplification in T225 cell culture flasks and cell factories. The cell culture temperature is 37°C ± 0.5°C. Multiple cell tubes taken from the same batch of the working cell bank at the same time are used only for the production of one batch of vaccine or virus seed after revival and amplification.

[0059] 3. Name and Source of Virus Seed

[0060] The virus seed for production is the SP-A strain of mumps virus that has been adapted and attenuated in human diploid cells KMB17 and belongs to the F genotype. The SP-A strain of mumps virus, after whole-genome sequencing, is of the F genotype (Genbank accession number: DQ 649478). The virus strain used this time is the 24th passage.

[0061] 4. Solution Preparation

[0062] 1) Taking the preparation of 1000 ml as an example, cell culture medium 01 (with 10% newborn bovine serum);

[0063] Table 1 Cell Culture Medium 01

[0064]

[0065] 2) Taking the preparation of 1000 ml as an example, cell culture medium 02 (with 5% newborn bovine serum);

[0066] Table 2 Cell Culture Medium 02

[0067]

[0068] 3) Taking the preparation of 1000 ml as an example, cell culture medium 03 (with 0% newborn bovine serum);

[0069] Table 3 Cell Culture Medium 03

[0070]

[0071] 4) 0.01 mol / L PBS buffer; in every 1000 ml of PBS solution, it contains: 8 g of NaCl, 0.2 g of KCl, 2.87 g of Na2HPO4·12H2O, and 0.2 g of KH2PO4, made up to 1000 ml with injection water, and the pH is 7.4;

[0072] 5) Taking the preparation of 1000 ml as an example, trypsin digestion solution;

[0073] Table 4 Trypsin

[0074]

[0075] 6) Virus maintenance medium (taking the preparation of 1000 ml as an example)

[0076] Table 5 Virus maintenance medium

[0077]

[0078] 5. A large-scale preparation method of mumps virus with low serum residue, the steps are as follows:

[0079] Step 1: Cell resuscitation and medium change: Resuscitate cells from the frozen state and inoculate them into 1 T225 culture flask, labeled as the 17th generation. The specific operation is to take out the KMB-17 cells from liquid nitrogen and immediately put them into the injection water at 37°C and shake back and forth continuously until the cell seeds are thawed. Add the cell seeds to a T225 cell culture flask containing 160 ml of cell culture medium 01 preheated to 37°C, cover the bottle cap, shake 5 times, place it in a constant temperature room at 37°C for cultivation, change the medium within 24 hours, discard the culture medium, and then add 160 ml of cell culture medium 01 preheated to 37°C, cover the bottle cap, and place it in a constant temperature room at 37°C for 5 days. The total number of cells during the whole cultivation process should not be less than 3.75×10 8 / cell factory.

[0080] Step 2: Cell passage X1: Take 1 T225 culture flask of the 17th generation and passage it to 4 T225 culture flasks, labeled as the 19th generation. The specific operation is to open the bottle cap of the T225 cell culture flask, pour out the old culture medium, add 0.01 mol / L PBS (pH 7.5) (50 ml ± 10 ml / bottle) to wash the cell surface, shake 6 times, and pour out the washing solution. Add trypsin digestion solution (9 ml ± 1 ml / bottle), shake 6 times to make the trypsin digestion solution infiltrate the cell surface, pour out the trypsin digestion solution, and add cell culture medium 01 within 10 minutes to start the suspension operation to terminate the action of trypsin.

[0081] The suspension operation of the cells in the cell culture flask is to add cell culture medium 01 to the cell culture flask (40 ml ± 10 ml / bottle), cover the lid, shake the cell culture flask vigorously 6 times to disperse the cells until there are no obvious clumps and make the cells mix evenly in the culture medium. After the suspension is completed, seed the cells according to the cell quantity and passage requirements at (160 ml ± 10 ml / bottle).

[0082] Step 3: Cell passage X2: Take 2 T225 culture flasks of the 19th generation and passage them to 8 T225 culture flasks, labeled as the 21st generation. (The cell passage method of the cell culture flask is the same as that in Step 2)

[0083] Step 4: Cell passage X3: Take 4 T225 culture flasks of the 21st generation and passage them to 16 T225 culture flasks, labeled as the 23rd generation. (The cell passage method of the cell culture flask is the same as that in Step 2)

[0084] Step Five, Cell Passage X4: Take 16 T225 culture flasks of the 23rd passage and passage them to 2 cell factories, labeled as the 24th passage. (The cell passage method for the culture flasks is the same as in Step Two).

[0085] Step Six, Cell Passage X5: Take 1 cell factory (CF) of the 24th passage and passage it to 4 cell factories, labeled as the 26th passage. The specific operation is as follows: Add 0.01 mol / L PBS (pH 7.5) (50 ml ± 10 ml / layer) to the 10-layer cell factory, shake the 10-layer cell factory 6 times to wash the cell surface, remove the washing solution, then add trypsin digestion solution (20 ml ± 5 ml / layer) to the 10-layer cell factory, shake the 10-layer cell factory 6 times to soak the cell surface with the trypsin digestion solution, remove the used trypsin digestion solution, and add cell culture medium 01 within 30 minutes to start the suspension operation to terminate the action of trypsin.

[0086] The suspension operation for the cell factory is to add cell culture medium 01 (75 ml ± 25 ml / layer) to the 10-layer cell factory, shake the 10-layer cell factory forcefully 6 times to disperse the cells until there are no obvious clumps and make the cells evenly mixed in the culture medium. After the suspension is completed, seed the cells according to the cell quantity and passage requirements at (200 ml ± 10 ml / layer CF).

[0087] Step Seven, Cell Passage X6: Take 2 cell factories of the 26th passage and passage them to 8 cell factories, labeled as the 28th passage. (The cell passage method for the cell factories is the same as in Step Six).

[0088] Step Eight, Cell Passage X7: Take 4 cell factories of the 28th passage and passage them to 16 cell factories, labeled as the 30th passage. (The cell passage method for the cell factories is the same as in Step Six).

[0089] Step Nine, Cell Passage X8: Take 12 cell factories of the 30th passage and passage them to 36 cell factories, labeled as the 32nd passage; among them, the culture medium used is cell culture medium 02 (the addition amount is 2000 ml / cell factory).

[0090] Step Ten, Virus Inoculation

[0091] The KMB17 cells were subcultured to passage 32 in a cell factory, cultured at 37 ± 0.5 °C for 4 days. Select a cell factory without contamination and in good growth condition, discard the old culture medium in the cell factory, then wash the cell surface with 0.01 mol / L PBS buffer (500 - 1000 ml / CF), shake 6 times, discard the washing solution, and then supplement with cell culture medium 03 (the addition amount is 1500 ml / cell factory), place it at 37 ± 0.5 °C for culture. When inoculating the virus on the 5th day of culture, discard the old culture medium in the cell factory, inoculate the mumps virus seed according to the MOI value of 0.02. Add it to the virus maintenance medium pre-warmed to 37 °C, mix well and then inoculate it into the cell factory (add 1500 ml of virus maintenance medium to each cell factory), place it at 37 °C for 8 days, and the harvested supernatant is the virus harvest fluid.

[0092] Comparative Example 1

[0093] Other methods were the same as in Example 1, the difference was only that during the whole culture process, only cell culture medium 01 was used to culture KMB-17 cells, and cell culture medium 02, cell culture medium 03 and virus maintenance medium were not used.

[0094] Experimental Example 1

[0095] Detect the residual bovine serum in the virus harvest fluid obtained in Example 1 and Comparative Example 1. Specifically, use the cygnus kit and perform experimental operations according to the instructions of the cygnus kit. The specific detection results are shown in Table 6.

[0096] Table 6 Detection Results of Residual Bovine Serum (Taking 1 Cell Factory as an Example)

[0097]

[0098] The opening of the cell factory will increase the risk of contamination, so the fewer the opening times, the lower the risk of contamination. In the comparative example: the number of openings: the first opening, discard the old culture medium, add 500 - 1000 ml of PBS solution to wash the cell surface; the second opening, discard the old PBS solution, repeat the above step; the third opening, discard the old PBS solution, and then add 1000 ml of PBS solution to soak for 10 min; the time-consuming is about 15 minutes.

[0099] In the example: the number of openings: the first opening, discard the old culture medium, add 1000 ml of PBS solution to wash the cell surface; the second opening, discard the old PBS solution, and then add cell culture medium 03; the time-consuming is about 5 minutes. Therefore, the culture method provided by the present invention can reduce the number of openings of the cell engineering, significantly reduce the operation time of the operating staff, and reduce the contamination risk of the cell factory.

[0100] Meanwhile, the method of the present invention gradually reduces the content of bovine serum in the culture medium, and finally reduces the content of bovine serum in the culture medium to 15.3 ng / dose, avoiding the potential influence of bovine serum.

[0101] This product is prepared by attenuated mumps virus vaccine strain (SP-A strain) placed in human diploid cell KMB 17 , cultured, harvested the virus solution, and freeze-dried after adding a suitable stabilizer. It is a cream-colored loose body and becomes an orange-red or light pink clear liquid after reconstitution. [Specification] 0.5 ml per bottle after reconstitution. The human dose per time is 0.5 ml.

[0102] It can be seen from the content recorded in Table 6 that the residual bovine serum content in the virus harvest fluid of the method of the present invention is only 15.3 ng / dose, while that of the traditional method is 48.5 ng / dose. The traditional method provided in Comparative Document 1 is close to the pharmacopoeia requirements and should not be higher than the standard of 50 ng / dose.

[0103] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A large-scale preparation method of mumps virus with low serum residue, characterized in that, It includes the following steps: (1) Digest the revived human diploid cells with protease, place them in cell culture medium 01, and perform subculture; (2) Place the human diploid cells obtained by subculture in step (1) in cell culture medium 02, and perform subculture; (3) Place the human diploid cells obtained by subculture in step (2) in cell culture medium 03 for culture; (4) Place the mumps virus seed stock in the human diploid cells obtained by culturing in step (3), then place it in virus maintenance medium for culture, and the harvested supernatant is the virus harvest fluid; The cell culture medium 01 is based on MEM solution and includes the following components: glutamine 1.8 - 2.2% (V / V), newborn bovine serum 9 - 11% (V / V), sodium bicarbonate 2 - 4% (V / V); The cell culture medium 02 is based on MEM solution and includes the following components: glutamine 1.8 - 2.2% (V / V), newborn bovine serum 3 - 5% (V / V), sodium bicarbonate 2 - 4% (V / V); The cell culture medium 03 is based on MEM solution and consists of the following components : glutamine 1.8 - 2.2% (V / V), sodium bicarbonate 2 - 4% (V / V); The virus maintenance medium is based on MEM solution and consists of the following components : glutamine 1.8 - 2.2% (V / V), sodium bicarbonate 4 - 6% (V / V).

2. The preparation method according to claim 1, characterized in that, The human diploid cells are KMB17 cells; the mumps virus seed stock is the F genotype mumps virus SP - A strain; the mass percentage concentration of glutamine is 2.5 - 3.5%; the mass percentage concentration of sodium bicarbonate is 6 - 7%.

3. The preparation method according to claim 1 or 2, characterized in that, The passage number of the human diploid cells in step (1) is the 16th - 28th passage.

4. The preparation method according to claim 1, characterized in that, The subculture of the human diploid cells in step (2) is up to the 32nd passage.

5. The preparation method according to claim 1, characterized in that, The culture time in step (3) is 3 - 5 d; the temperature is 36.5 - 37.5 °C.

6. The preparation method according to claim 1, wherein, The culture time in step (4) is 7 - 9 d; the temperature is 36.5 - 37.5 °C; in step (4), the mumps virus seed stock is inoculated according to the MOI value of 0.02 - 0.

2.

7. The preparation method according to claim 1, characterized in that, In step (2), when placing the cells in cell culture medium 02, the cells need to be washed with buffer first; in step (3), when placing the cells in cell culture medium 03, the cells need to be washed with buffer first.

8. The preparation method according to claim 7, characterized in that, The buffer is prepared from the following components in mass concentration: NaCl 7 - 9 g / L, KCl 0.1 - 0.3 g / L, Na2HPO4·12H2O 2 - 3 g / L, and KH2PO4 0.1 - 0.3 g / L, pH 7.3 - 7.

5.

9. The virus harvest fluid prepared by the preparation method according to any one of claims 1 - 8.

10. The application of the virus harvest fluid according to claim 9 in the preparation of mumps live attenuated vaccine.

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