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Virus liquid concentration method

A virus solution and virus technology, applied in the field of virus culture and concentration of virus solution, can solve the problems of inability to obtain virus, long separation time, discomfort of virus particles, etc., and achieve a simple and easy concentration method, maintain virus infection ability, and improve virus infection. effect of titer

Active Publication Date: 2018-11-13
苏州良辰生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is required that the titer of the indicator virus must reach 106TCID50 or more, but different viruses have different proliferation effects on cells, and conventional proliferation and culture cannot obtain high-titer viruses, so necessary concentration methods must be adopted to increase the virus titer
[0004] As a method of increasing the concentration of viruses in a solution and concentrating viruses, there are physical methods such as ultracentrifugation, but this method requires expensive equipment, a long separation time, and requires a lot of labor in the operation
There are also chemical methods, such as the method of concentrating the virus by precipitating the virus with ammonium sulfate, polyethylene glycol, etc., but in these methods, because the reagents used or other components for precipitation are harmful to the infected cells, there are obstacles that hinder subsequent operations. Hidden dangers and tedious work such as purification must be carried out after virus precipitation
In addition, there is a method of using antibodies to concentrate the virion itself, but this method inhibits the function of the protein on the surface of the virus, which may affect the infectivity of the virus. For example, when separating the virion from the antibody, strong acidic conditions must be used (pH2~3) for elution, which has the potential to cause discomfort to virus particles

Method used

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  • Virus liquid concentration method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Cell subculture: Take out 1 tube of frozen ST cells from liquid nitrogen, thaw quickly in a 37°C water bath, use a sterile pipette to draw 1.0ml of cell suspension into a 15ml sterile centrifuge tube, and add drop by drop MEM medium containing 10wt% FBS to 5-15mL, pipet several times, mix evenly, centrifuge immediately (3000rpm, 5min), and discard the supernatant. Then add 10-20mL of 10wt% FBS MEM medium, pipette several times, mix well, transfer to T25 culture bottle with 10wt% FBS MEM medium, place at 37°C, 5% CO 2 Cultivate in an incubator for 48h to 72h, and when the cells cover a monolayer, set aside.

[0023] 2. Subculture of the virus: Take out 1 tube of frozen PPV virus from the -70°C refrigerator (working virus bank), redissolve in a 37°C water bath, dilute 10 times with MEM medium without FBS, and then inoculate 1mL in In the cell bottle that has been covered with a monolayer of cells, store at 37°C, 5% CO 2 The incubator was adsorbed for 1 hour, and the ...

Embodiment 2

[0029] 1. Cell subculture: Take out 1 tube of frozen LLC-MK2 cells from liquid nitrogen, thaw quickly in a 37°C water bath, use a sterile pipette to draw 1.0ml of cell suspension into a 15ml sterile centrifuge tube, drop by drop Add dropwise DMEM medium containing 10wt% FBS to 5-15mL, pipette several times, mix well, centrifuge immediately (3000rpm, 5min), and discard the supernatant. Then add 10-20 mL of DMEM medium with 10wt% FBS, pipette several times, mix well, transfer to T25 culture bottle with DMEM medium with 10wt% FBS, place at 37°C, 5% CO 2 Cultivate in an incubator for 24h to 48h, and when the cells cover a single layer, set aside.

[0030] 2. Subculture of the virus: take out a tube of frozen ReoV-3 virus from the -70°C refrigerator (working virus bank), redissolve it in a 37°C water bath, dilute it 10 times with MEM medium without FBS, and then pipette 1mL Inoculate in a cell bottle that has been filled with a monolayer of cells, store at 37°C, 5% CO 2 The incub...

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Abstract

The invention relates to a virus liquid concentration method. Biological membranes are placed into a vessel, virus liquid is added into the vessel according to the quantity that 18-25ml of virus liquid is added into 1g of biological membranes, the virus liquid is freeze-dried after adsorption for 1-3 hours at the temperature of 2-8 DEG C and then redissolved by an MEM culture medium containing 1-3wt% of fetal calf serum, the biological membranes are cut into pieces and then homogenized, biological membrane fragments are centrifugally removed, and supernate is filtered to obtain concentrated virus liquid. The concentration method is simple and easy, the concentrated virus liquid keeps virus infection capacity, virus titer (not lower than 107.0 TCID50 / 0.1ml) is improved, the separated virusliquid can be obtained, and the virus liquid concentration method has a wide application prospect in virus removal / inactivation verification.

Description

technical field [0001] The invention belongs to virus culture technology in the field of microbe application, and in particular relates to a method for concentrating virus liquid. Background technique [0002] Biopharmaceutical products such as monoclonal antibodies, recombinant proteins, vaccines, blood derivatives, and animal products carry the risk of transmitting infectious viruses because the source material may itself be contaminated with viruses or virus-like particles. Furthermore, the production process of biopharmaceutical products is susceptible to viral contamination from external sources. Manufacturers of biopharmaceutical products therefore need to include adequate virus removal steps in their manufacturing processes to ensure that their products are free of contaminating viruses. [0003] The most commonly used viral assays to quantify viral contaminants in viral clearance studies are the tissue culture limiting dose 50% (TCID50), plaque forming unit (PFU) an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02
CPCC12N7/00
Inventor 吴娟缪汝娉鲍大为
Owner 苏州良辰生物医药科技有限公司
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