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Application of bacteriophage in verification of biological product virus removal process

A technology of biological products and bacteriophage, which is applied in the direction of microorganism-based methods, measurement/inspection of microorganisms, microorganisms, etc., can solve the problems of long time for registration and declaration of biological products, a large amount of time and economic costs, and high requirements for experimental operation skills, so as to save The effect of economic cost and labor cost, shortening the detection time, and saving experimental materials

Pending Publication Date: 2020-03-24
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

[0004] In the existing biosafety level two laboratories, virus amplification is limited by the limitations of virus infection (relying on cell infection, the number of cells per unit volume is constant, resulting in a constant number of virus amplification), and the titer of the virus is generally Not high, up to 10 7 PFU / mL, and then purified and concentrated up to 10 8 PFU / mL, and purification and concentration generally require ultra-high-speed centrifugation and chromatography steps. This step requires high instrument cost, long time, and high requirements for personnel's experimental operation skills
Therefore, cultivating these viruses, purifying the viruses, and increasing the virus titer currently require a lot of time and economic costs.
[0005] In addition, the detection time of these indicator viruses currently used generally takes 7 days to 21 days, which makes the registration and declaration of biological products take a long time

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the preparation of purifying phage MS2

[0021] Bacteriophage MS2 (ATCC 15597-B1) and host bacteria: Escherichia coli (ATCC 13706) were purchased from ATCC (American Type Culture Collection). On this basis, phage amplification is carried out, and the specific steps are as follows: 1. Add working library Escherichia coli to the pre-configured LB culture medium and cultivate for 16-24 hours; 2. Mix the MS2 stock solution with the cultivated Escherichia coli Mix 1:3 evenly and let it stand for 15 minutes to infect; 3. Connect the mixed solution to LB culture medium, and incubate on a shaker at 37°C, 200RPM, for 6 hours; 4. Add inducer (IPTG, 1mM) at 37°C, 200RPM, 18-24 hours; centrifuge at 5.4°C, 4000RPM, 5 minutes, discard the supernatant, and collect the bacteria; 6. Add PBS buffer according to the ratio of 1:20, ultrasonic (300W, working time 10s, interval 10s, cycle 40 times ); 7. Add Benzonase nuclease to the solution after ultrasonic lysis and incubate...

Embodiment 2

[0024] Embodiment 2, the virus removal validation process of removing MS2 phage in bovine serum albumin (BSA) by nanofiltration.

[0025] 1. Take commercially available BSA standard solution (0.48mg / ml) as a sample, take 250ml, and filter through a 0.22um filter to remove impurities.

[0026] 2. According to the ratio of 1:1000, take 0.25ml of purified MS2 solution (titer 10 14 PFU / ml), added to the above BSA solution, mixed well and then sampled.

[0027] 3. Take the nano-membrane (model: Viresolve NFP) produced by Millipore Company, the surface area is 3.5cm 2 , after wetting and pressurizing to test the integrity of the membrane, it is installed on the nano-membrane filter.

[0028] 4. The above-mentioned BSA solution mixed with MS2 is subjected to nano-membrane filtration at a pressure of 2 psi.

[0029] 5. Take samples at the time points of filtering 50mL, 100mL, 150mL, 200mL, 250mL and MS2 stock solution respectively.

[0030] 6. Take out the double-layer agar plate ...

Embodiment 3

[0033] Embodiment 3, DEAE anion exchange resin removes the virus removal validation process of MS2 phage in bovine serum albumin (BSA).

[0034] 1. Column packing

[0035] Take 12mL of the settled DEAE resin and add it to the plexiglass chromatography column, and assemble it into the AKTA protein purification system, and wash it with 50mL deionized water to balance.

[0036] 2. Packing cleaning

[0037] Wash the column with 50 mL of 0.5 M NaOH solution at a rate of 1.0 mL / min.

[0038] 3. Ion exchange chromatography equilibrium

[0039] Equilibrate the chromatography column with 120mL equilibration buffer at a flow rate of 2.0mL / min. After the A280 baseline is stable, set the UV value to zero.

[0040] 4. Sample loading

[0041]Take 100mL of commercially available BSA standard solution (0.48mg / mL), add the purified MS2 solution at a ratio of 1:10000, mix well, take a sample and mark it as sample 1 to be tested, and load the remaining samples at a flow rate of 2.0mL / min. W...

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Abstract

The invention relates to application of bacteriophage in verification of a biological product virus removal process. According to the invention, bacteriophage is used as an indicator virus in verification of a virus removal process, and the indicator virus has the advantages of small size, high titer and simple purification; the detection requirements of biological product verification laws and regulations are met by adding a small amount of bacteriophage; experimental materials are saved; the detection time is short; the process verification speed is accelerated; and the economic cost and thelabor cost are saved.

Description

technical field [0001] The invention belongs to the field of biological product safety, and in particular relates to the application of bacteriophage in the process verification of biological product virus removal. Background technique [0002] At present, domestic and foreign biopharmaceutical fields must go through virus removal / inactivation process verification before applying for marketing. Biological products derived from cell culture and other sources have the risk of virus contamination, which will lead to serious consequences in clinical practice. The possible source of contamination is Due to the original cell line itself, it may also come from the foreign virus accidentally brought in during the production process. In order to ensure the safety of biotechnology products, the virus needs to be eliminated and inactivated during the production process to reduce the number of virus-like particles in each dosage unit. content to 10 -6 "Statistical Issues in the Evaluat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/22C12R1/92
CPCC12Q1/22C12Q1/04
Inventor 汪涛严娜曹忆周洁殷梦琪
Owner 苏州良辰生物医药科技有限公司
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