CN114082224A - Purification method suitable for large-scale plasmid DNA production - Google Patents
Purification method suitable for large-scale plasmid DNA production Download PDFInfo
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- CN114082224A CN114082224A CN202010856171.XA CN202010856171A CN114082224A CN 114082224 A CN114082224 A CN 114082224A CN 202010856171 A CN202010856171 A CN 202010856171A CN 114082224 A CN114082224 A CN 114082224A
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- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 23
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/363—Anion-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3819—Affinity chromatography of the nucleic acid-nucleic acid binding protein type
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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. Firstly, anion exchange chromatography medium is taken as a stationary phase to carry out ion exchange chromatography to remove impurities such as RNA, endotoxin, escherichia coli host protein and the like, and plasmids are reserved; then affinity chromatography is carried out to remove the adsorbed supercoiled plasmid and other trace impurities to obtain the purified DNA plasmid. The method combines ion exchange chromatography and affinity chromatography for plasmid purification, and has short time and low cost compared with the traditional purification process; the gel filtration step is not needed, the volume of a sample treated by direct chromatography is not limited by the volume of a column and is only related to the amount of plasmids to be purified, the feed liquid with the same volume is purified, the consumption of a chromatography medium is only 1/10 or less of that of the traditional gel filtration chromatography medium, the diameter of the required chromatography column is smaller when the process is amplified, and the cost can be saved.
Description
Technical Field
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.
Background
The purification method of high-purity plasmid DNA commonly used in the market at present is three-step chromatography: removing RNA by gel filtration chromatography, selectively adsorbing supercoiled plasmid by affinity chromatography, and removing endotoxin and other impurities by ion exchange chromatography.
The sample loading volume in the gel filtration chromatography step involved in the method is constrained by the column volume of the chromatography and the viscosity of the feed liquid, and the sample loading volume is at most 30 percent of the column volume. When the process is further amplified, the required chromatographic column has larger diameter, more chromatographic medium consumption and higher cost. And the gel filtration chromatographic column generally has the phenomena of low flow rate and easy collapse in the using process, the production process consumes relatively long time, and the repacking frequency of the chromatographic column is relatively high. Due to principle limitation, gel filtration chromatography can only complete plasmid capture, cannot remove impurities larger than plasmid molecules, and cannot effectively reduce the levels of impurities such as endotoxin, escherichia coli host protein, genome and the like. After the second step of chromatography, the plasmid purity is relatively low, and the plasmid needs to be purified by the third step of ion exchange chromatography, but the plasmid DNA yield in the step is relatively low, so that the total yield of the process is relatively low.
Therefore, there is a need to develop a purification process for plasmid DNA that is low in cost and takes a short time.
Disclosure of Invention
In view of the above, the present invention aims to provide an anion exchange chromatography medium for plasmid purification.
Separation principle of anion exchange chromatography medium: in a specific pH environment, plasmids are negatively charged and can be bound to positively charged anion exchange chromatography media by charge interaction. By changing the pH value or the conductance of the buffer solution and utilizing the difference of different molecular charges, the binding can be respectively released for elution, thereby realizing the separation effect. DEAE is diethylaminoethyl, is one of ligands carried by anion exchange chromatography media, has relatively weak charge, and is suitable for plasmid purification.
The chromatography medium ligand comprises agarose, polystyrene, cross-linked polymethacrylate polymer and the like, and is combined with different ligands according to different process requirements. The medium for conventional plasmid purification is a gel filtration chromatography medium, and separation is achieved by using the size of molecules. For purification, the fractions are collected as molecules larger than a specific size, with a lower resolution than ion exchange chromatography.
The anion exchange chromatography medium is: the ligand is DEAE (diethylaminoethyl), and the ligand is cross-linked polymethacrylate polymer beads.
Further, in the present invention,the density of the ligand is 0.29-0.35mmol cl-1A/ml filler.
The invention aims to further provide an application of the DEAE anion exchange chromatography medium in plasmid purification. The literature shows that DEAE has only application in protein purification at present, and has no relevant application in plasmid purification.
The invention also aims to provide a method for purifying a mixture containing plasmids.
The method is to carry out ion exchange chromatography by taking the anion exchange chromatography medium as a stationary phase. The anion exchange chromatography medium is specifically: the ligand is DEAE (diethylaminoethyl), and the ligand is polystyrene rigid microspheres.
Further, the density of the ligand is 0.29-0.35mmol cl-1A/ml filler.
Further, the mixture includes RNA, plasmids, endotoxins, e.
Further, the method specifically comprises the following steps: loading the mixture onto an ion exchange chromatography column; after the loading is finished, the chromatographic column is washed by using the equilibrium buffer solution for 3-5 column volumes until the absorption value at 275-285nm is reduced to be close to the baseline level, and the peaks in the loading and column washing processes are not collected. Elution was performed with elution buffer, 3-5 column volumes were washed, the peak was collected, and collection was stopped when the absorbance at 275-285nm dropped to a near baseline level.
Further, the balance buffer solution is 20-150mM MOPs and/or Tris and/or HEPES and/or PIPES, and the pH value is 6.5-7.5.
Further, the elution buffer is 20-150mM MOPs and/or Tris and/or HEPES and/or PIPES, and the pH value is 6.5-7.5.
Further, the chromatographic column of the ion exchange chromatography is selected from the chromatographic columns with the diameters of 26mm, 50mm, 100mm, 200mm and 600mm and the column packing height of 100-200 mm.
Specifically, the ion exchange chromatography is used for separating RNA, plasmid, endotoxin and escherichia coli host protein, firstly removing the RNA, the endotoxin, the escherichia coli genome and the escherichia coli host protein, and retaining the plasmid to achieve the purpose of purification.
The invention aims to provide a large-scale plasmid DNA purification method.
The purification method comprises the following steps: (1) treating the raw materials by the method for purifying the mixture and collecting eluent; (2) and replacing the eluent by buffer solution, and then processing by affinity chromatography to obtain purified plasmid DNA.
Further, the raw material in the step (1) is pretreated, and the pretreatment specifically comprises the following steps: performing alkali lysis on initial feed liquid to obtain feed liquid, concentrating the feed liquid by a tangential flow ultrafiltration system through a die pack with the aperture of 100 + 500kD or a hollow fiber column to obtain concentrated feed liquid, and replacing the concentrated feed liquid by using an ion exchange equilibrium buffer solution with the pH value of 6.5-7.5, wherein the replacement multiple is 3-5 times of the volume of the concentrated feed liquid.
Further, the buffer solution in the step (2) is 20-150mM MOPs and/or Tris and/or HEPES and/or PIPES, and the pH value is 7.0-8.0.
Further, the exchange solution of the ion exchange equilibrium buffer solution is 20-150mM MOPs, Tris, HEPES and PIPES.
Further, the purified plasmid DNA in the step (2) is subjected to post-treatment, wherein the post-treatment specifically comprises the following steps: and (3) concentrating and replacing the elution peak collection component by using a die pack or a hollow fiber column with the pore diameter of 30-300kD for the purified plasmid DNA, replacing the buffer solution with a low-conductivity phosphate buffer solution or TE buffer solution, and then sterilizing and filtering the plasmid DNA solution after replacement to obtain the medicinal plasmid DNA.
Further, the chromatography medium of the affinity chromatography in the step (2) is plasmid select Xtra.
Specifically, the affinity chromatography of step (2) removes mainly adsorbed supercoiled plasmid and other trace impurities.
The invention aims to provide a purification method suitable for large-scale plasmid DNA production, which comprises the following steps:
(1) loading the raw material to an ion exchange chromatography column; after the loading is finished, the chromatographic column is washed by using the equilibrium buffer solution for 3-5 column volumes until the absorption value at 275-285nm is reduced to be close to the baseline level, and the peaks in the loading and column washing processes are not collected. Elution was performed with elution buffer, 3-5 column volumes were washed, the peak was collected, and collection was stopped when the absorbance at 275-285nm dropped to a near baseline level.
(2) And replacing the eluent by buffer solution, and then processing by affinity chromatography to obtain purified plasmid DNA.
(3) And (3) concentrating and replacing the elution peak collection component by using a die pack or a hollow fiber column with the pore diameter of 30-300kD for the purified plasmid DNA, replacing the buffer solution with a low-conductivity phosphate buffer solution or TE buffer solution, and then sterilizing and filtering the plasmid DNA solution after replacement to obtain the medicinal plasmid DNA.
Further, the equilibration buffer and elution buffer are 20-150mM MOPs and/or Tris and/or HEPES and/or PIPES, and the pH value is 7.0-8.0.
Specifically, the ion exchange chromatography is used for separating RNA, plasmid, endotoxin and escherichia coli host protein, RNA, endotoxin escherichia coli genome and escherichia coli host protein are removed firstly, and plasmid is reserved to achieve the purpose of purification.
Further, the chromatography medium of the affinity chromatography in the step (2) is plasmid select Xtra.
The invention has the beneficial effects that:
the invention combines ion exchange chromatography and affinity chromatography for plasmid purification, and has short time and low cost compared with the traditional purification process.
The invention does not need a gel filtration step, the volume of a sample treated by direct chromatography is not limited by the volume of a column, and is only related to the amount of plasmids to be purified, the feed liquid with the same volume is purified, the consumption of a chromatography medium is only 1/10 or less of that of the traditional gel filtration chromatography medium, the diameter of the required chromatography column is smaller when the process is amplified, and the cost can be saved.
Drawings
FIG. 1: ion exchange chromatography.
FIG. 2: affinity chromatography.
FIG. 3: purification process sample agarose gel electrophoresis picture.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
EXAMPLE 1 bacterial liquid lysis
After the engineering bacteria are fermented at high density, the bacteria are collected by high-speed centrifugation and then are suspended in a heavy suspension buffer solution; or collecting concentrated bacteria liquid through a hollow fiber column and replacing the concentrated bacteria liquid with a heavy suspension buffer solution; adding lysis solution into the bacterial suspension, and gently and uniformly mixing for lysis; after cracking, adding neutralizing liquid and mixing homogeneously to stop reaction.
EXAMPLE 2 concentration of bacterial liquid lysate
After the lysate obtained in example 1 is clarified by a depth filter or a hollow fiber column, a tangential flow ultrafiltration system is used for concentrating the lysate by a 100-one 500kD pore size module or a hollow fiber column, and then the solution is exchanged by ion exchange equilibrium buffer solution with the pH value of 6.5-7.5, wherein the exchange solution multiple is 3-5 times of the volume of the concentrated feed solution.
Example 3 ion exchange chromatography
Equilibrating the ion exchange chromatography column with ion exchange equilibration buffer for 3-5 column volumes in an AKTA chromatography system from GE; loading the clarified concentrated solution obtained in the step 2 to an ion exchange chromatography column; after the sample loading is finished, washing the chromatographic column by using an equilibrium buffer solution for washing 3-5 column volumes until the absorption value at 280nm is reduced to be close to the baseline level, and not collecting peaks in the sample loading and column washing processes;
eluting with elution buffer solution, washing 3-5 column volumes, collecting the peak, and stopping collection until the absorption value at 280nm is reduced to near baseline level; washing the chromatographic column with 0.5M NaOH solution for 3-5 column volumes until the absorption value at 280nm is reduced to near baseline level, and collecting no washing solution. There are 3 major peaks in the chromatogram, wherein the first peak is mainly composed of RNA (band 4 in FIG. 3), the second peak is mainly composed of plasmid DNA (band 5 in FIG. 3), and the third peak is other impurities. The results are shown in FIG. 1.
Example 4 affinity chromatography
Example 3 the purified sample was purified on an affinity column using PlasmidSelect Xtra as the chromatography medium. And collecting elution peaks in the chromatographic process. In a tangential flow ultrafiltration system, the elution peak-collecting components are subjected to a concentrate exchange using a die pack or hollow fiber column having a pore size of 30-300kD, and the buffer is replaced with a low conductance phosphate buffer or TE buffer.
The results are shown in FIG. 2: the first peak is the unbound or loosely bound component, the major component is the open circular structure of the plasmid (band 9 in FIG. 3), and the second peak is the supercoiled structure of the plasmid (band 10 in FIG. 3).
EXAMPLE 5 Filter Sterilization and examination of the purification results
And sterilizing and filtering the plasmid solution after liquid replacement, subpackaging, storing at low temperature (2-8 ℃) or freezing at (-20 ℃ -80 ℃), and warehousing after passing inspection to obtain the medicinal plasmid DNA.
The test results are shown in fig. 3: 3. 4, 5 sequentially carrying out ion exchange chromatography sample loading, penetrating and washing column liquid (a first peak of an ion exchange chromatography spectrum) in the sample loading stage, and eluting and collecting components (a second peak of the ion exchange chromatography spectrum); 8. 9 and 10, sequentially carrying out affinity chromatography sample loading, penetrating and washing column liquid (a first peak of an affinity chromatography), and eluting and collecting components (a second peak of the affinity chromatography).
The results of the purification comparison of the present invention with the conventional scheme are shown in table 1:
table 1 comparison of purification results of the present invention and the prior art
TABLE 2 comparison of time consuming and wearing of the purification of the present invention with the prior art
| Technique of | Cost/time consuming |
| Prior Art | 260 yuan/mg, and takes 28 h/batch |
| The present invention | 50 yuan/mg, which takes 16 h/batch |
TABLE 3 Chinese and English comparison table of each impurity and sample
| English | Chinese character |
| pDNA | Plasmids |
| scDNA | Supercoiled plasmid |
| Endo | Bacterial endotoxins |
| HCP | Host proteins |
| gDNA | Genome |
| RNA | Ribonucleic acid |
| Recovery | Yield of |
As can be seen from tables 1, 2 and 3, the contents of endotoxin and E.coli host protein in the purified plasmid of the present invention are lower than those in the prior art, and the total process yield is 77% higher than that in the prior art by 69%. The purification time is shortened by 12 hours, the purification cost is less than 1/5 of the original process, the yield is higher than that of the original process, and the purification effect is not inferior to that of the original process. The effect of the invention for purifying plasmid is superior to that of the background technology, and the invention has strong scalability, lower cost and shorter time consumption.
Example 6 comparison of materials of the prior art and the present invention
The plasmid was produced at 100mg, and the inventive ratio was compared with the prior art-required material, as shown in Table 3 below.
TABLE 4 materials required for the prior art production of 100mg plasmid
| Step (ii) of | Kind of medium | Number of media |
| First step of | Gel filtration chromatography | 1.3L |
| Second step of | Affinity chromatography | 50ml |
| The third step | Ion exchange chromatography | 50ml |
TABLE 5 materials required for the production of 100mg of plasmid according to the invention
| Step (ii) of | Kind of medium | Number of media |
| First step of | Ion exchange chromatography (DEAE) | 100ml |
| Second step of | Affinity chromatography | 50ml |
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (10)
1. An anion exchange chromatography medium for plasmid purification, wherein said anion exchange chromatography medium is: the ligand is DEAE (diethylaminoethyl), and the ligand is cross-linked polymethacrylate polymer beads.
2. The anion exchange chromatography media of claim 1, wherein the ligand density is 0.29-0.35mmol cl-1A/ml filler.
The use of a DEAE anion exchange chromatography medium for the purification of plasmids.
4. A method for separating a mixture containing plasmids by ion exchange chromatography using the anion exchange chromatography medium of claim 1 or 2 as a stationary phase.
5. The method of claim 4, wherein the mixture comprises RNA, plasmids, endotoxins, E.coli genome, and E.coli host proteins.
6. The method according to claim 4 or 5, characterized in that the method is in particular: loading the mixture onto an ion exchange chromatography column; after the sample loading is finished, washing the chromatographic column by using an equilibrium buffer solution for washing 3-5 column volumes until the absorption value at 275-285nm is reduced to the level close to the baseline, and not collecting the peak in the process of loading and washing the column; elution was performed with elution buffer, 3-5 column volumes were washed, the peak was collected, and collection was stopped when the absorbance at 275-285nm dropped to a near baseline level.
7. The method according to claim 4, wherein the equilibration buffer is 20-150mM MOPs or/and Tris or/and HEPES or/and PIPES, pH 6.5-7.5.
8. A method for the purification of large scale plasmid DNA, characterized in that said purification method comprises the steps of: (1) treating the starting material by the method of any one of claims 4 to 6 to collect the eluate; (2) and replacing the eluent by buffer solution, and then processing by affinity chromatography to obtain purified plasmid DNA.
9. The purification process according to claim 8, wherein the buffer in step (2) is 20 to 150mM MOPs or/and Tris or/and HEPES or/and PIPES, and the pH value is 7.0 to 8.0.
10. The purification method according to claim 8 or 9, wherein the purified plasmid DNA of step (2) is subjected to a post-treatment, in particular: and (3) concentrating and replacing the elution peak collection component by using a die pack or a hollow fiber column with the pore diameter of 30-300kD for the purified plasmid DNA, replacing the buffer solution with a low-conductivity phosphate buffer solution or TE buffer solution, and then sterilizing and filtering the plasmid DNA solution after replacement to obtain the medicinal plasmid DNA.
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| CN115612684A (en) * | 2022-11-01 | 2023-01-17 | 北京启辰生生物科技有限公司 | GMP-grade plasmid DNA large-scale simplified production method |
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| CN111304193A (en) * | 2020-02-28 | 2020-06-19 | 恒瑞源正（上海）生物科技有限公司 | Method for large-scale rapid purification of plasmid DNA |
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| CN1243542A (en) * | 1997-01-10 | 2000-02-02 | 罗切诊断学有限公司 | Method for purifying DNA in cross-flow centrifuge |
| CN101139591A (en) * | 2006-09-06 | 2008-03-12 | 中国人民解放军第二军医大学 | Small molecule RNA of shark liver and use thereof |
| CN103930546A (en) * | 2011-09-26 | 2014-07-16 | 凯杰有限公司 | Rapid method for isolating extracellular nucleic acids |
| CN103657617A (en) * | 2012-09-14 | 2014-03-26 | 丁少峰 | Chromatographic column |
| CN111304193A (en) * | 2020-02-28 | 2020-06-19 | 恒瑞源正（上海）生物科技有限公司 | Method for large-scale rapid purification of plasmid DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115612684A (en) * | 2022-11-01 | 2023-01-17 | 北京启辰生生物科技有限公司 | GMP-grade plasmid DNA large-scale simplified production method |
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