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Large-scale preparation technology of endotoxin-free plasmid

A technology of endotoxin and quality, applied in the biological field, can solve problems such as inapplicability

Inactive Publication Date: 2018-06-12
NANJING IASO BIOTHERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, commercialized plasmid purification kits are not suitable for the preparation of sufficient quantities of gram-level and above plasmid products

Method used

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  • Large-scale preparation technology of endotoxin-free plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Small-scale preparation of ten grams or less of endotoxin-free lentiviral plasmid

[0025] Prepare lentivirus purification plasmids with a scale of 10 grams or less. Shake flasks are selected for cell fermentation, and the cells are collected by a centrifuge. The clarified lysate after lysis is centrifuged in a refrigerated high-speed centrifuge, and 1ml or 5ml of anion exchange is used for column chromatography. Prepacked columns.

[0026] 1. Shake flask fermentation

[0027] Take a recombinant strain from the seed bank, streak it on the resistance plate, and expand the single colony in 5mL liquid medium containing the corresponding antibiotic for 8h, and then inoculate it into a 1L container with a ratio of 1:1000. In a 3L shake flask of the culture medium, shake culture at 180-220rpm at 37°C for 16h.

[0028] 2. Cell collection

[0029] Refrigerated high-speed centrifuge at 8000g 4°C for 3min to collect the bacterial cells, and weigh the wet weight of t...

Embodiment 2

[0037] Example 2: Preparation of endotoxin-free lentiviral plasmids on a scale of more than ten grams

[0038] For the preparation of purified lentivirus plasmids of more than 10 grams, shake flask fermentation is limited by its yield, so it is necessary to use a fermenter for fermentation. The fermenter can increase the bacterial cell production per unit medium on a large scale, and can be scaled up in parallel; For clarification, you can choose a refrigerated high-speed centrifuge, which is time-consuming, or you can choose TFF. Compared with the two, TFF is more suitable for large-scale production, but the cost of consumables is higher; the selection of column volume can refer to the binding amount of 2g / ml. choose.

[0039] 1. Fermentation tank fermentation bacteria

[0040] Take a recombinant strain from the seed bank, streak it on the resistance plate, expand the single colony in the liquid medium containing the corresponding antibiotic, and then inoculate it into a 100...

Embodiment 3

[0050] Embodiment 3: Culture medium is optimized to improve plasmid yield

[0051] Comparing LB, SB, TSB, TB four kinds of media under the same culture conditions, the wet weight of bacteria and plasmid yield per unit volume of culture medium, the medium with high plasmid yield was selected out of them. Shake the bacteria in the flask under the same conditions for the following four culture media, take 25ml of the bacterial liquid each, and extract the plasmid with an endotoxin-free plasmid extraction kit.

[0052] LB medium formula: 1% (W / V) tryptone, 0.5% (W / V) yeast extract, 1% (W / V) NaCl.

[0053] SB medium formula: 3.5% (W / V) tryptone, 2.0% (W / V) yeast extract, 0.5% (W / V) NaCl, 0.5% (V / V) 1M NaOH.

[0054] TSB medium formula: 1.5% (W / V) tryptone, 0.5% (W / V) soybean peptone, 0.5% (W / V) NaCl, adjust the pH to 7.2±0.2.

[0055] TB medium formula: 1.2% (W / V) tryptone, 2.4% (W / V) yeast extract, 0.4% (V / V) glycerol, 17mM KH2PO4, 72mM K2HPO4.

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Abstract

The invention belongs to the technical field of biology, and specifically discloses a large-scale extracting technology of an endotoxin-free plasmid. The large-scale extracting technology is characterized by comprising the following steps: strain fermentation, collection of bacterial cells, splitting of bacteria, solid-liquid separation of a bacteria splitting product, collection of a liquid part,and purification of a liquid part of the bacteria splitting product, wherein the purification comprises the following steps: adding an endotoxin removing buffer solution, removing impurities and other plasmids through low-temperature treatment and ionization chromatography, and collecting target plasmids, wherein the pH (Potential of Hydrogen) of the endotoxin removing buffer solution is 7.0, andthe endotoxin removing buffer solution is prepared from 3-(N-morpholine)propanesulfonic acid, sodium chloride and polyethylene glycol octylphenol ether. A method disclosed by the invention does not use RNase A and protease K which are derived from animals and phenol and chloroform which have toxic and side effects on human bodies and animals; all indexes can achieve or approach the quality standard of FDA (U.S Food and Drug Administration) in related field; meanwhile, linear amplification can be realized; an important technological means is provided for industrial production of eukaryote products and development of gene therapy industry.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a plasmid extraction technology, in particular to a large amount of endotoxin-free plasmid extraction technology. Background technique [0002] Plasmids are small, double-stranded circular DNA molecules that carry genetic information outside of chromosomes. Because plasmids are self-replicating, a cell can contain multiple copies of plasmids. In the past few decades, plasmids have become the most important tools in the fields of genetics and molecular biology, especially in recent years, they have become indispensable tools in the fields of gene therapy and gene vaccines. At present, the application of plasmid DNA in clinical research such as gene therapy and gene vaccine is more and more important. Subsequently, the requirements for plasmid production technology in the therapeutic field are getting higher and higher. It requires not only high yield, but also high purity and impurity...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 杨永坤孟广荣
Owner NANJING IASO BIOTHERAPEUTICS CO LTD
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