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CN-122012488-A - Method for purifying plasmid DNA based on membrane chromatography

CN122012488ACN 122012488 ACN122012488 ACN 122012488ACN-122012488-A

Abstract

The invention relates to the field of biotechnology, in particular to a method for purifying plasmid DNA based on membrane chromatography. The method uses a membrane chromatography technology, does not need a column loading experiment, only needs 1-2 hours at one time for purifying plasmids with a faster working flow rate, and has the advantages of high DNA recovery rate of 73 percent, high supercoiled DNA recovery rate of 97 percent and high supercoiled DNA proportion of 95 percent.

Inventors

  • Chen Qunyang
  • HUANG YINGMIN
  • YE ZHICHENG
  • WANG JUAN

Assignees

  • 云舟生物科技(广州)股份有限公司

Dates

Publication Date
20260512
Application Date
20241111

Claims (10)

  1. 1. A method for purifying plasmid DNA based on membrane chromatography, comprising: purifying the plasmid DNA solution by an affinity chromatography membrane to obtain purified plasmid DNA; the purification comprises loading, washing impurities and eluting.
  2. 2. The purification method according to claim 1, wherein the ligand of the affinity chromatography membrane is 2-mercaptopyridine.
  3. 3. The method according to claim 1, wherein the impurity-washed solution contains 1.8M to 1.9M (NH 4 ) 2 SO 4 , 100mM Tris-HCl,10mM EDTA and water, and has a pH of 7.5.+ -. 0.3.
  4. 4. The method of claim 1, wherein the eluted solution contains 0.39m to 1.51m (NH 4 ) 2 SO 4 +0.6M~1.61M NaCl,100mM Tris-HCl,10mM EDTA and water, pH 7.5±0.3).
  5. 5. The method according to claim 4, wherein the eluted solution contains 1.44M (NH 4 ) 2 SO 4 +0.6M NaCl,100mM Tris-HCl,10mM EDTA and water, pH 7.5.+ -. 0.3).
  6. 6. The method of purifying according to claim 1, wherein the loading comprises the steps of equilibration, loading and re-equilibration; The balanced solution comprises 1.8-1.9M (NH 4 ) 2 SO 4 , 100mM Tris-HCl,10mM EDTA and water, and the pH value is 7.5+/-0.3; The sample contains plasmid DNA 5-500 ng/. Mu.L, 1.8-1.9M (NH 4 ) 2 SO 4 , 100mM Tris-HCl,10mM EDTA and water, and the pH value is 7.5+/-0.3; the re-equilibrated solution comprises 1.8M-1.9M (NH 4 ) 2 SO 4 , 100mM Tris-HCl,10mM EDTA and water, and the pH value is 7.5 + -0.3.
  7. 7. The purification method according to any one of claims 1 to 6, wherein the flow rate of the solution in the purification step is 10MV/min; In the sample loading step, the balanced solution is 60-80 MV; in the sample loading step, the sample loading solution is 50-150 MV; In the sample loading step, the re-balanced solution is 80-120 MV; In the impurity washing step, the solution is 80-120 MV; In the elution step, the solution is 100-150 MV.
  8. 8. The method according to any one of claims 1 to 6, further comprising the step of washing after the eluting, wherein the washing is performed by using a washing liquid 1 and a washing liquid 2, and then the washing is performed by using a preservation liquid; The cleaning solution 1 is an aqueous solution containing 1MNaOH and 1M NaCl; The cleaning solution 2 comprises 1.8-1.9M (NH 4 ) 2 SO 4 , 100mM Tris-HCl,10mM EDTA and water, and the pH value is 7.5+/-0.3; The preservation solution is 0.1M NaOH aqueous solution.
  9. 9. A purification reagent for plasmid DNA based on membrane chromatography, which comprises a wash solution and an eluent; The impurity washing liquid contains 1.8-1.9M (NH 4 ) 2 SO 4 , 100mM Tris-HCl,10mM EDTA and water, and the pH value is 7.5+/-0.3; the eluent contains 0.39M-1.51M (NH 4 ) 2 SO 4 +0.6M~1.61MNaCl,100mM Tris-HCl,10mM EDTA and water, and the pH is 7.5+/-0.3.
  10. 10. The purification reagent of claim 9, further comprising a equilibration solution and a loading solvent: The balancing solution comprises 1.8-1.9M (NH 4 ) 2 SO 4 , 100mM Tris-HCl,10mM EDTA and water, and the pH value is 7.5+/-0.3); The loading solvent comprises 1.8-1.9M (NH 4 )2SO 4 , 100mM Tris-HCl,10mM EDTA and water, and the pH value is 7.5+/-0.3).

Description

Method for purifying plasmid DNA based on membrane chromatography Technical Field The invention relates to the field of biotechnology, in particular to a method for purifying plasmid DNA based on membrane chromatography. Background Plasmid DNA is a key raw material for many gene therapies, such as raw materials for the production of viral vectors, as templates for in vitro transcription of mRNA, as an effective vector for the therapy itself, etc., and the trend in gene therapy complexity has led to greater plasmid DNA requirements. The plasmid purification process is a technique that separates plasmid DNA from host DNA, RNA, host proteins, endotoxins, and non-supercoiled plasmid conformations. High quality, high purity plasmid DNA is a critical component in cell and gene therapy processes, plasmid purity can seriously affect transfection efficiency, and if the plasmid is impure, impurities contained therein can produce cytotoxicity or seriously affect the formation of transfection complexes, so that purification of plasmid DNA is a critical step in the production process. In plasmid production, E.coli is generally selected as a host cell, and the cell is lysed after fermentation culture to obtain a plasmid. Various plasmid forms such as supercoiled plasmid DNA, open-loop DNA, linear DNA, plasmid DNA aggregate and the like can exist in the cleaved feed liquid, and impurities such as host proteins, host DNA, RNA, endotoxin and the like are accompanied. These impurities can be removed by chromatographic purification processes. The existing plasmid purification process mainly comprises the following steps: (1) The classical three-step process (molecular sieve + hydrophobic chromatography + anion chromatography), the first step uses gel filtration chromatography, based on plasmid DNA and RNA molecular weight difference to realize separation, the step can obtain about 80% supercoiled plasmid purity, and the yield exceeds 90%, and at the same time the buffer solution replacement is completed, so that the next step of plasmid affinity chromatography operation, in the plasmid affinity chromatography step, the filler using 2-mercaptopyridine as ligand is used, the main purpose is to remove non-closed-loop DNA, and realize high-efficiency separation of supercoiled plasmid, through this step, the supercoiled plasmid purity can be raised to above 85%, and finally, the anion exchange chromatography is used for fine purification, and the step is mainly aimed at removing residual endotoxin and trace impurities. (2) Based on a simple improvement of the classical three-step method, the removal of RNA is carried out by using calcium chloride or ammonium sulfate precipitation instead of molecular sieve, and other steps are similar to the three-step method. (3) Two-step process based on complex mode Capto Core 700 and hydrophobic chromatography or plasmid affinity chromatography. Capto Core 700 is a composite mode filler, the outer surface of the filler microsphere is an inert shell, the hole is internally provided with a strongly adsorbed octylamine group, the molecular exclusion of more than 700KD is outside, and impurities with smaller molecular weight are combined with the octylamine group in the hole. In general, plasmid DNA is separated in a flow-through mode because the molecules of the plasmid DNA are larger and are discharged outside the filler microsphere, RNA, host protein, host nucleic acid and endotoxin enter the inside of the filler microsphere to be combined with an octyl amine group, so that the purpose of separation is achieved, and the second step of chromatography can adopt hydrophobic chromatography or plasmid affinity chromatography, so that the supercoiled proportion of the plasmid is improved and the plasmid is precise. (4) Anion exchange chromatography and hydrophobic chromatography, wherein the anion exchange chromatography can utilize the difference of charge properties of plasmids and impurities to remove impurities such as RNA, endotoxin and the like in plasmid samples, and the hydrophobic chromatography or plasmid affinity chromatography can remove non-closed-loop plasmids to carry out precise purification on the samples. As can be seen, in the prior art, in order to obtain plasmid DNA with higher purity, a multi-step chromatography process is often required, the required process time is longer, the cost is higher, and the operation is more complicated, so that a process for rapidly purifying the plasmid needs to be developed. The membrane chromatography is a high-efficiency separation technology, and can couple ligands on microporous filter membranes made of PES, PP and other materials, so that the ligands have binding capacity. Depending on the coupling ligand, membrane chromatography currently includes anion exchange chromatography membranes, cation exchange chromatography membranes, hydrophobic chromatography membranes, and the like. Heretofore, membrane chromatography has been used in the proce