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Method for producing gene therapy plasmids by adopting starvation fermentation process, and fermentation culture medium

A fermentation medium and fermentation process technology, which is applied in the field of gene therapy plasmid and fermentation medium production by starvation fermentation process, can solve problems such as low yield, and achieve the effects of reducing acid production, reducing HCP residual content and reducing HCP residual content.

Pending Publication Date: 2020-04-24
无锡生基医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This patent adopts this fermentation medium, and the yield of plasmid fermentation (5L) reaches 180mg / L~220mg / L, which is relatively low.

Method used

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  • Method for producing gene therapy plasmids by adopting starvation fermentation process, and fermentation culture medium
  • Method for producing gene therapy plasmids by adopting starvation fermentation process, and fermentation culture medium
  • Method for producing gene therapy plasmids by adopting starvation fermentation process, and fermentation culture medium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The basic steps of embodiment 1 disposable fully enclosed plasmid fermentation process

[0034] 1. The main equipment of one-time fermentation process

[0035] device name brand Bioreactor Eppendorf Aseptic tube machine Sartorius Aseptic tube sealer Sartorius

[0036] 2. List of main consumables for one-time fermentation process

[0037]

[0038]

[0039] 3. One-time fermentation process control parameters

[0040] 1) Fermentation tank preparation: calibrate the pH electrode, insert the pH electrode, DO electrode and cooling pipeline into the corresponding position of the disposable tank, install the feeding and inoculation pipeline and seal the outlet end with a plug. After the fermentation medium is prepared, put the tank body into a pulse vacuum sterilizer at 121.0°C for 20 minutes to sterilize. After sterilization, install the pipelines for the intake, exhaust and cooling of the fermenter, connect the feeding and alkali ...

Embodiment 2

[0044] The study of embodiment 2 starvation fermentation technology influences on plasmid HCP

[0045] 1. Use fermentation medium 1 formula: each 1L fermentation medium contains 10g of soybean peptone, 5g of yeast powder, 0g of ammonium sulfate, 7.7g of disodium hydrogen phosphate heptahydrate, 1.5g of potassium dihydrogen phosphate, 16g of glycerol, and magnesium sulfate 0.6g, defoamer 0.3g.

[0046] Feed medium formula, each 1L feed medium contains 100g of peptone, 50g of yeast powder, 150g of glycerol, and 1g of antifoaming agent.

[0047] Feed medium formula, each 1L feed medium contains 100g of peptone, 50g of yeast powder, 150g of glycerol, and 1g of antifoaming agent.

[0048] Feeding lye formula, every 1L of feeding lye contains 120g of sodium hydroxide.

[0049] 2. Starvation fermentation method: correlate DO and speed control, control DO ≥ 20%, minimum speed 200rpm, maximum 1200rpm, open DO automatic control. Define the inoculation time as 0 hour, start feeding wh...

Embodiment 3

[0058] Embodiment 3: Escherichia coli host protein residue detection method

[0059] 1. Standard product preparation: the detection kit is E.coli HCP enzyme-linked immunosorbent assay kit (CYGNUS company), and the standard products 0, 1, 3, 12, 40, 100 ng / mL that come with the kit are taken out and equilibrated to room temperature.

[0060] 2. Dilution of the test sample: prepare 1×PBST washing solution, and use the sample diluent (CYGNUS company) to dilute the sample gradually to the standard curve range of 1-100ng / mL.

[0061] 3. Plate layout: Take out the coated plate, add 25 μL / well of the standard product and the test product respectively, and make 2 duplicate wells for each.

[0062] 4. Add enzyme-labeled antibody: Add 100 μL / well of HRP-labeled antibody to all wells, seal the plate with membrane, place in a constant temperature shaker, set the temperature at 28°C, and incubate at 600 rpm for 90 minutes.

[0063] 5. Wash the plate: Discard the sample solution in each we...

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Abstract

The invention discloses a method for producing gene therapy plasmids by adopting a starvation fermentation process. The method comprises the following steps: (1) preparing a fermentation tank: inserting a disposable pH electrode, a disposable DO electrode and a cooling pipeline into corresponding positions of a disposable tank body; sterilizing the disposable tank body after the fermentation culture medium is prepared; completing the connection of each pipeline of the fermentation tank, setting the temperature of a temperature control system (TCU) and operating; (2) setting parameters; (3) carrying out inoculation; and (4) controlling starvation fermentation. The method provided by the invention combines a starvation fermentation process and a one-time totally-enclosed process, and can significantly reduce the residual content of bacterial host protein HCP in a plasmid product. The invention further discloses the fermentation culture medium for the method, and the fermentation yield ofplasmids can be effectively increased.

Description

technical field [0001] The invention belongs to a fermentation method for gene therapy plasmids in the field of biotechnology, and in particular relates to a method for producing gene therapy plasmids using a starvation fermentation process and a safe and high-yield fermentation medium. Background technique [0002] Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate diseases caused by gene defects and abnormalities, so as to achieve therapeutic purposes. Gene therapy has become an effective treatment for many major diseases, and has made significant progress in the treatment of hemophilia, sickle cell disease, blindness, a variety of severe genetic neurodegenerative diseases, and multiple cancers of the bone marrow and lymph nodes (Gene therapy comes of age, 2018, Science). At present, the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) have approved 6 gene therapy products: 2 chimeric a...

Claims

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Application Information

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IPC IPC(8): C12Q3/00C12P1/04C12N15/63C12R1/19
CPCC12Q3/00C12P1/04C12N15/63C12R2001/19C12N1/205
Inventor 曾杰梁焯曹曦姚树元辛晨浩
Owner 无锡生基医药科技有限公司