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Purification method suitable for large-scale plasmid DNA production

A purification method and plasmid technology, applied in biochemical equipment and methods, DNA preparation, chemical instruments and methods, etc., can solve the problems of high cost, easy collapse, and large diameter of chromatographic columns, and achieve low cost, small diameter, cost saving effect

Pending Publication Date: 2022-02-25
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the process is further enlarged, the diameter of the required chromatography column is larger, the amount of chromatography medium is larger, and the cost is higher
In addition, gel filtration chromatography columns generally have low flow rate and easy collapse during use, the production process takes a relatively long time, and the frequency of repacking the chromatography column is relatively high
Due to the limitations of the principle, gel filtration chromatography can only capture the plasmid, and cannot remove impurities larger than the plasmid molecule, nor can it effectively reduce the level of impurities such as endotoxin, E. coli host protein, and genome
After the second step of chromatography, the purity of the plasmid is also relatively low, and it needs to be purified by the third step of ion exchange chromatography, but the yield of plasmid DNA in this step is relatively low, so that the overall yield of the process is relatively low

Method used

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  • Purification method suitable for large-scale plasmid DNA production
  • Purification method suitable for large-scale plasmid DNA production
  • Purification method suitable for large-scale plasmid DNA production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Bacteria solution lysing

[0043] After high-density fermentation of engineering bacteria, the bacteria are collected by high-speed centrifugation and resuspended in the resuspension buffer; or the concentrated bacterial solution is collected through a hollow fiber column and replaced with a resuspension buffer; the lysate is added to the bacterial suspension and mixed gently After the lysis is completed, add the neutralizing solution and mix evenly to stop the reaction.

Embodiment 2

[0044] Embodiment 2 bacterial liquid lysates concentrate

[0045] After clarifying the lysate obtained in Example 1 with a depth filter or a hollow fiber column, use a tangential flow ultrafiltration system to concentrate through a mold bag or a hollow fiber column with a pore size of 100-500kD, and then use a pH value of 6.5-7.5 The ion-exchange equilibrium buffer is changed, and the change ratio is 3-5 times the volume of the concentrated feed solution.

Embodiment 3

[0046] Embodiment 3 ion exchange chromatography

[0047] In the AKTA chromatographic system of GE Company, the ion-exchange chromatography column is balanced with ion-exchange equilibration buffer, and 3-5 column volumes are balanced; the concentrated solution after clarification in Example 2 is loaded to the ion-exchange chromatography column; After the end, rinse the chromatography column with equilibration buffer for 3-5 column volumes until the absorbance at 280nm drops to near the baseline level, and do not collect the peaks during loading and column washing;

[0048]Elute with elution buffer, wash 3-5 column volumes, collect the peak, stop collecting when the absorbance at 280nm drops to near baseline level; wash the chromatography column with 0.5M NaOH, wash 3-5 columns Volume, until the absorbance at 280nm drops to near the baseline level, do not collect the flushing solution. There are 3 main peaks in the chromatogram, wherein the main component of the first peak is ...

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Abstract

The invention belongs to the field of biological separation and purification, and particularly relates to a purification method suitable for large-scale plasmid DNA production. The method comprises the following steps: carrying out ion exchange chromatography by taking an anion exchange chromatography medium as a stationary phase to remove impurities such as RNA, endotoxin and Escherichia coli host protein, and retaining plasmids; and carrying out affinity chromatography to remove and adsorb the superhelix plasmids and other trace impurities so as to obtain purified DNA plasmids. According to the method, ion exchange chromatography and affinity chromatography are combined for plasmid purification, and compared with a traditional purification process, the time is short, and the cost is low; a gel filtration step is not needed, the volume of a sample subjected to direct chromatography treatment is not limited by the column volume and is only related to the quantity of plasmids to be purified, feed liquid with the same volume is purified, the dosage of a chromatography medium is only 1 / 10 or less of that of a traditional gel filtration chromatography medium, the diameter of a chromatography column needed during process amplification is small, and the cost can be saved.

Description

technical field [0001] The invention belongs to the field of biological separation and purification, and in particular relates to a purification method suitable for large-scale plasmid DNA production. Background technique [0002] At present, the commonly used purification method of high-purity plasmid DNA in the market is three-step chromatography: gel filtration chromatography to remove RNA, affinity chromatography for supercoiled plasmid selective adsorption, and ion exchange chromatography to remove endotoxin and other impurities. [0003] In the gel filtration chromatography step involved in the method, the sample loading volume is restricted by the chromatography column volume and the feed liquid viscosity, and the sample loading volume is at most 30% of the column volume. When the process is further enlarged, the diameter of the required chromatography column is larger, the amount of chromatography medium is larger, and the cost is higher. Moreover, gel filtration ch...

Claims

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Application Information

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IPC IPC(8): B01D15/36B01D15/38C12N15/10
CPCB01D15/363B01D15/3819C12N15/101C12Q2523/308
Inventor 王戬任秋雨
Owner CHONGQING PRECISION BIOTECH CO LTD
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