CN114164204A

CN114164204A - Method for purifying plasmid

Info

Publication number: CN114164204A
Application number: CN202010948587.4A
Authority: CN
Inventors: 李彦林; 陈明曦; 任江涛; 贺小宏; 王延宾; 韩露
Original assignee: Jiangsu Puzhu Biomedical Technology Co ltd
Current assignee: Nanjing Bioheng Biotech Co Ltd
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2022-03-11

Abstract

The invention provides a method for purifying plasmids by fixed column chromatography, which comprises the following steps: (1) cracking the Escherichia coli culture containing the plasmid to be extracted to obtain a cracking solution; (2) carrying out chromatography by using a first fixed column to obtain a crude purified solution; (3) and (5) carrying out chromatography on the crude purification solution by using a second fixed column to obtain purified plasmid. The invention improves the purification efficiency by optimizing the process conditions such as conductivity, the proportion of elution buffer solution, the working flow rate of the fixed column, the dilution multiple of the sample and the like.

Description

Method for purifying plasmid

Technical Field

The invention belongs to the field of biological pharmacy. More particularly, the present invention relates to a method of plasmid purification, which is particularly suitable for plasmid preparation in pilot plant or industrial production.

Background

Plasmids are an extrachromosomal genetic element that occurs widely in nature. At present, plasmids are widely used in gene transfer operation as genetic information carriers. In particular, in recent years, with the rapid development of advanced therapeutic methods (including gene therapy and cell therapy), the production of plasmids has been gradually shifted from laboratories to industrial mass production.

Generally, in the laboratory, plasmids can be extracted by a plasmid extraction kit, but this method has a small single extraction amount. Therefore, in industrial production, extraction and purification are generally performed by methods such as alkaline lysis, purification, ultra-separation and concentration. Wherein, the purification step mainly adopts a column chromatography method.

Conventional column chromatography methods mainly use chromatography packing materials of various ligands, and gel particles are generally selected as matrixes in the manufacturing process of the packing materials. However, the purification filler has the problem of low loading in the industrial production of plasmids, for example, in the technology of the plasmid purification platform of GE healthcare, the loading of plasmids on the plasmid select Xtra filler is only 2 mg/ml. In addition, the packed column is usually operated at a flow rate of 450cm/h, and a high flow rate cannot be achieved, which also results in a long process time (about 1.5 hours is generally required for purifying 200ml of lysate).

The use of fixed columns can overcome the problems of low loading and low flow rate. The fixed column is a novel chromatographic column which is manufactured by crosslinking a filler ligand and a fixed membrane matrix according to a fixed volume and has the characteristics of high ligand density and high pressure resistance.

Therefore, a method for purification using fixed columns, which is optimized in terms of conditions, is required to improve the purification efficiency.

Disclosure of Invention

The invention provides a method for purifying plasmids, which comprises the following steps: (1) cracking the Escherichia coli culture containing the plasmid to be extracted to obtain a cracking solution; (2) carrying out chromatography by using a first fixed column to obtain a crude purified solution; (3) and (5) carrying out chromatography on the crude purification solution by using a second fixed column to obtain purified plasmid.

In one embodiment, the method of the present invention further comprises: after the step (1), the lysate is clarified to obtain a lysis supernatant, and the lysis supernatant is subjected to chromatography by using a first fixed column. Methods of performing clarification are known to those skilled in the art, for example by centrifugation, filtration through a filter, and the like.

In one embodiment, the first fixed columns are anion exchange fixed columns, preferably DEAE fixed columns.

In one embodiment, the second fixed columns are hydrophobic fixed columns, preferably C4 hydrophobic fixed columns.

In one embodiment, the loading conductivity of the first fixed column is from 35 to 45mS/cm, preferably from 36 to 42.5mS/cm, more preferably 40 mS/cm.

Equilibration buffers are typically used to bring the stationary column to equilibrium prior to chromatography, thereby minimizing the effects of the external environment on plasmid purification. If necessary, a certain proportion of equilibration buffer may be used for elution of plasmids. In one embodiment, the first stationary column employs a first equilibration buffer comprising 5-15mM EDTA and 10-75mM Tris (pH7.2), preferably 8-12mM EDTA and 25-60mM Tris (pH7.2), more preferably 10mM EDTA and 50mM Tris (pH 7.2). In one embodiment, the second stationary column employs a second equilibration buffer comprising 2-4M ammonium sulfate, 5-15mM EDTA and 10-75mM Tris (pH7.2), preferably 2.5-3.5M ammonium sulfate, 8-12mM EDTA and 25-60mM Tris (pH7.2), more preferably 3M ammonium sulfate, 10mM EDTA and 50mM Tris (pH 7.2).

The elution buffer is mainly used for eluting components adsorbed on the fixed column, such as plasmids, RNA and the like. The composition of the elution buffer is generally different depending on the components to be eluted. In one embodiment, the first fixed column uses a first elution buffer comprising 0.5-2M NaCl, 5-15mM EDTA, and 10-75mM Tris (pH7.2), preferably 0.75-1.5M NaCl, 8-12mM EDTA, and 25-60mM Tris (pH7.2), more preferably 1M NaCl, 10mM EDTA, and 50mM Tris (pH 7.2). In one embodiment, the second elution buffer used for the second stationary column comprises 5-15mM EDTA and 10-75mM Tris (pH7.2), preferably 8-12mM EDTA and 25-60mM Tris (pH7.2), more preferably 10mM EDTA and 50mM Tris (pH 7.2).

The ratio of the elution buffer is the ratio of the elution buffer used for elution of the plasmid. For example, the ratio of the elution buffer solution X means that when the plasmid is eluted, the elution buffer solution at the ratio X and the equilibration buffer solution at the ratio 1-X are used. In one embodiment, the proportion of elution buffer in the first fixed column is 80% to 100%. In one embodiment, the proportion of elution buffer in the second stationary column is 40% -60%.

The working flow rate refers to the flow rate of the equilibration buffer and the elution buffer when the fixed column is used for chromatography. In one embodiment, the first fixed column has a working flow rate of 50 ml/min. In one embodiment, the second fixed column has an operating flow rate of 8 to 50 ml/min.

In one embodiment, the second fixed column is diluted 1-fold with sample.

The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

Drawings

FIG. 1: gel electrophoresis images of eluted samples at different conductivities when chromatographed using the first fixed column. M: marker; 4-1 and 4-2 represent the lysis supernatant and the penetrant, respectively, during the chromatography of the sample diluted 4 times; 5-1, 5-2 and 5-3 represent lysis supernatant, penetration liquid and eluent 1 in the chromatography process of the sample diluted by 5 times respectively; 6-1, 6-2 and 6-3 represent lysis supernatant, penetration liquid and eluent 1 in the chromatography process of the sample diluted by 6 times respectively; 7-1, 7-2, 7-3 and 7-4 represent lysis supernatant, penetration liquid and eluent 1 and eluent 2, respectively, in the chromatographic process of the sample diluted by 7 times; 8-1, 8-2, 8-3 and 8-4 represent the lysis supernatant, the permeate and the eluent 1 and the eluent 2, respectively, during the chromatography of the sample diluted 8 times. The RNA bands are indicated in boxes.

FIG. 2: gel electrophoresis images of eluted samples at different dilutions of the sample when chromatographed using the second stationary column. M: marker; 1-1, 1-2 and 1-3 represent crude purified liquid, permeate liquid and eluent with sample dilution factor of 0.5 times, respectively; 2-1, 2-2 and 2-3 represent crude purified liquid, penetration liquid and eluent with sample dilution factor of 1.0 times respectively; 3-1, 3-2 and 3-3 represent the crude purified liquid, the permeate liquid and the eluent with the sample dilution ratio of 2.0 times, respectively; 4-1, 4-2 and 4-3 represent the crude purification solution, the permeation solution and the elution solution, respectively, at a sample dilution ratio of 3.0 times. The open circular plasmid band is shown by the arrow.

Detailed Description

The composition of the solution used in this example is shown in table 1 below.

TABLE 1

Name (R)	Composition of solution
		Resuspension buffer	TE buffer, pH8.0
Lysis buffer	0.2M NaOH + 2% SDS solution
		Neutralization buffer	3M Potassium acetate buffer, pH5.5
First equilibration buffer	TE buffer, pH7.2
		First elution buffer	TE buffer, pH7.2+1.0M NaCl
Second equilibration buffer	3M (NH4)2SO4+ TE buffer, pH7.2
		Second elutionBuffer solution	TE buffer, pH7.2

Note: the TE buffer was 10mM EDTA +50mM Tris.

Example 1 optimization of first fixed column purification conditions

Lysis of bacterial cultures by alkaline lysis: and (3) resuspending the Escherichia coli culture containing the plasmid to be purified uniformly by using a resuspension buffer solution with the pH of 8.0, then adding a lysis buffer solution with an equal proportion, and after 5 minutes, adding a neutralization buffer solution with an equal proportion for neutralization to obtain a lysate. The lysate was centrifuged at 4200rpm for 20min at 4 ℃ and filtered through a 1 micron filter to obtain 3.7L of lysate supernatant.

(1) Optimizing sample loading conductivity

The purpose of the first fixed column is mainly to remove RNA contaminants.

100ml of the lysis supernatant was diluted 4 times, 5 times, 6 times, 7 times, and 8 times with ultrapure water, respectively, to adjust the conductivity, and then subjected to chromatography through a first fixed column (i.e., DEAE fixed column) as follows: firstly, balancing 10 times of column volume of a fixed column by adopting a first balance buffer solution, then loading a sample at a working flow rate of 50ml/min, and collecting a solution flowing out of the first fixed column at the moment, namely a penetration solution; after the sample is completely loaded, balancing the chromatographic column by 10 times of the column volume by adopting a first balance buffer solution; then, the first fixed column is continuously and linearly eluted by adopting a first elution buffer solution (the proportion is gradually increased from 0 to 100 percent), and parts with the ultraviolet absorption value being more than 100mAU are sequentially collected, namely eluent 1 and eluent 2. Finally, the column is equilibrated again with the first equilibration buffer until the experiment is complete.

The concentrations of the permeate, eluent 1 and eluent 2, and the purity of the resulting plasmid were determined and the results are shown in table 2 below.

TABLE 2 purification results at different conductivities

Dilution factor	4	5	6	7	8
						Conductivity (mS/cm)	51.63	42.23	36.40	32.36	28.59
Penetration liquid volume (ml)	320	400	480	560	640
						Concentration of penetration liquid (ng/. mu.l)	26.3	24.8	20.0	15.5	10.3
Volume of eluent 1 (ml)	NA	10	7.5	24	48
						Eluent 1 concentration (ng/. mu.l)	NA	52.9	70.2	42.3	81.0
Eluent 2 vol (ml)	NA	NA	NA	7.5	8.0
						Eluent 2 concentration (ng/. mu.l)	NA	NA	NA	73.2	76.2
Purity (%)	/	82.51	76.90	74.11	74.06

Furthermore, 10. mu.l of the permeation solution, eluent 1 and eluent 2 were subjected to gel electrophoresis, and the results are shown in FIG. 1.

As can be seen from table 2 and fig. 1, when the conductivity was too high (diluted 4-fold), an eluate containing the plasmid could not be obtained, indicating that the plasmid was not adsorbed onto the DEAE column; when the conductivity was too low (7-8 fold dilution), the eluate 1 contained a higher concentration of RNA (see 7-3 and 8-3 of FIG. 1), indicating that at this conductivity RNA would also adsorb to the fixed column, which would have a severe adverse effect on the plasmid loading. When the dilution factor is 5-6 times, only the plasmid is adsorbed to the fixed column, and the RNA is basically retained in the penetration liquid, so that the loading capacity of the chromatographic column is retained, and the purification pressure of the chromatographic column is reduced. And the plasmid purity in the crude purification solution obtained at this time was the highest.

Thus, the optimum conductivity for the first fixed column purification is 35-45mS/cm, preferably 36-42.5 mS/cm.

(2) Optimizing the working flow rate and the ratio of elution buffer

200ml of the lysis supernatant was diluted 5.5 times with ultrapure water, adjusted to a conductivity of 40mS/cm, and then chromatographed through a DEAE fixed column, as follows: firstly, balancing 10 times of column volume of a fixed column by using a first balance buffer solution, then sampling at a working flow rate of 20ml/min or 50ml/min, and collecting a solution flowing out of the first fixed column, namely a penetration solution; after the sample is completely loaded, balancing the chromatographic column by 10 times of the column volume by adopting a first balance buffer solution; then, the first fixed column is washed by 40%, 60%, 80% and 100% of first elution buffer solution, and the part with the ultraviolet absorption value larger than 100mAU is collected, namely the eluent. Finally, the column is equilibrated again with the first equilibration buffer until the experiment is complete.

The concentrations of the permeate and the eluate, as well as the purity of the resulting plasmid, were determined and the results are shown in table 3 below.

TABLE 3 purification results for different flow rates and ratios of elution buffer

As can be seen from Table 3, the ratios of elution buffers of 40% and 60% failed to elute the objective plasmid. At a flow rate of 20ml/min, 80% of the elution buffer can achieve a relatively good elution effect, and at a flow rate of 50ml/min, 80% -100% of the elution buffer can achieve a relatively good elution effect.

The optimum elution buffer flow rate for the first fixed column purification is 50ml/min, the elution buffer proportion is 80-100%, considering that the higher the flow rate, the shorter the time required for the purification process.

(3) Purification effect after optimization of conditions

200ml of the lysis supernatant was diluted 5.5 times with ultrapure water, adjusted to a conductivity of 40mS/cm, and then chromatographed through a DEAE fixed column, as follows: firstly, balancing 10 times of column volume of a fixed column by adopting a first balance buffer solution, then loading a sample at a working flow rate of 50ml/min, and collecting a solution flowing out of the first fixed column at the moment, namely a penetration solution; after the sample is completely loaded, balancing the chromatographic column by 10 times of the column volume by adopting a first balance buffer solution; then, the first fixed column is washed by 100% of first elution buffer solution, and the part with the ultraviolet absorption value larger than 100mAU is collected, namely the eluent. Finally, the column is equilibrated again with the first equilibration buffer until the experiment is complete. The experiment was repeated twice. Each experiment took about 14 minutes.

The concentrations of the permeate and the eluate and the purity of the resulting plasmid were determined and the loading of the fixation column was calculated and the results are shown in table 4 below.

TABLE 4 purification Effect under optimized conditions

Sample (I)	Experiment 1	Experiment 2
			Sample concentration (ng/. mu.l)	20	16.9
Sample size (ml)	1000	1000
			Concentration of penetration liquid (ng/. mu.l)	16.7	16.8
Penetration liquid volume (ml)	1000	1000
			Concentration of eluent (ng/. mu.l)	220.8	216.2
Volume of eluent (ml)	28	28
			Load capacity (mg/ml)	4	4
Purity (%)	93.05	93.91

As can be seen from Table 4, under the condition of optimized conductivity, elution buffer flow rate and elution buffer ratio, the loading capacity of the fixed column can reach 4mg/ml, the purity of the purified plasmid can reach about 93 percent, and the purpose of purifying the plasmid is realized.

Example 2 optimization of second fixed column purification conditions

The primary purpose of the second stationary column is to remove the open circular plasmid.

The E.coli culture was lysed according to the method described in example 1, and purified using DEAE fixed columns under the conditions that the conductivity was 40mS/cm, the working flow rate of the first elution buffer was 50ml/min, and the proportion of the first elution buffer was 100%, to obtain 60ml of a crude purification solution as a sample to be applied to the second fixed column.

(1) Optimization of sample dilution factor

50ml of the crude purified solution was diluted 0.5-fold, 1-fold, 2-fold, 3-fold with 4M ammonium sulfate, and then chromatographed through a second fixed column (i.e., a C4 hydrophobic fixed column) as follows: firstly, balancing 10 times of column volume of a fixed column by using a second balance buffer solution, then sampling at a second working flow rate of 50ml/min, and collecting a solution flowing out of the fixed column at the moment, namely a penetration solution; after the sample is completely loaded, balancing the chromatographic column by 10 times of the column volume by adopting a second balance buffer solution; and then, continuously and linearly eluting the second fixed column by using a second elution buffer solution (the proportion is gradually increased from 0 to 100 percent), and collecting the part with the ultraviolet absorption value of more than 100mAU, namely the eluent. Finally, the column was equilibrated again with a second equilibration buffer until the experiment was completed.

The concentrations of the eluents and the purities of the plasmids obtained were measured, and the results are shown in Table 5 below.

TABLE 5 purification Effect at different sample dilution times

Dilution factor	0.5 times of	1 times of	2 times of	3 times of
					Sample concentration (ng/. mu.l)	32.7	24.5	16.4	12.3
Sample volume (ml)	60	80	120	160
					Concentration of eluent (ng/. mu.l)	61.7	71.7	73.2	72.1
Volume of eluent (ml)	16.5	17.5	20	20
					Purity (%)	69.19	77.73	61.54	63.69
Yield (%). I	36.5	49.6	45.8	46.7

Yield% (% eluted sample concentration × eluted sample volume × purity)/(loaded sample volume × loaded sample concentration).

As can be seen from Table 4, with the increase of dilution factor, the purity and yield of plasmid appeared to increase first and then decrease.

Furthermore, 10. mu.l of the crude purified solution, the permeate and the eluate were subjected to gel electrophoresis, and the results are shown in FIG. 2.

As can be seen from FIG. 2, in the case of the 0.5-fold and 1-fold dilutions, a partially open circular plasmid (as indicated by the arrow) was present in the transudates, whereas in the transudates of the 2-fold and 3-fold dilutions, no bands of open circular plasmid were present. This indicates that, at higher dilution times, the open circular plasmid cannot pass out at the loading stage, which increases the risk of the open circular plasmid entering the eluate, thereby affecting product purity.

Thus, the optimal sample dilution factor for the second fixed column purification is 1-fold.

(2) Optimizing the ratio of the working flow rate and the second buffer

200ml of the crude purification solution was diluted 1-fold with 4M ammonium sulfate and then chromatographed through a C4 fixed column as follows: firstly, balancing the 10 times of column volume of a fixed column by using a second balance buffer solution, then loading the sample at the working flow rate of 8ml/min or 50ml/min, and collecting the solution flowing out of the fixed column at the moment, namely the penetration solution; after the sample is completely loaded, balancing the chromatographic column by 10 times of the column volume by adopting a second balance buffer solution; then, the chromatographic column is washed by 40 percent or 60 percent of second elution buffer solution, and the part with the ultraviolet absorption value more than 100mAU is collected, namely the eluent. Finally, the column was equilibrated again with a second equilibration buffer until the experiment was completed.

The concentrations of the eluents and the purities of the plasmids obtained were measured, and the results are shown in Table 6 below.

TABLE 6 purification results for different flow rates and ratios of elution buffer

As can be seen from table 6, there was no significant difference in the purity of the obtained plasmid at different working flow rates and different ratios of elution buffer, which had no significant effect on the purification effect of the second stationary column.

Therefore, the optimal working flow rate for the second fixed column purification is 8-50ml/min, and the elution buffer ratio is 40-60%.

(3) Purification effect after optimization of conditions

200ml of the crude purification solution was diluted 1-fold with 4M ammonium sulfate and then chromatographed through a C4 fixed column as follows: firstly, balancing 10 times of column volume of a fixed column by using a second balance buffer solution, then loading a sample at a working flow rate of 50ml/min, and collecting a solution flowing out of the fixed column at the moment, namely a penetration solution; after the sample is completely loaded, balancing the chromatographic column by 10 times of the column volume by adopting a second balance buffer solution; then, the chromatographic column is washed by 60 percent of second elution buffer solution, and the part with the ultraviolet absorption value more than 100mAU is collected, namely the eluent. Finally, the column was equilibrated again with a second equilibration buffer until the experiment was completed. Three experiments were repeated. Each experiment took about 28 minutes.

The concentration of the eluate and the purity of the obtained plasmid were measured, and the yield was calculated, and the results are shown in table 7 below.

TABLE 7 purification Effect under optimized conditions

	Experiment 1	Experiment 2	Experiment 3
				Sample concentration (ng/ul)	257	289.1	313
Sample volume (ml)	47	50	50
				Concentration of eluent (ng/ul)	201.4	212.3	234.2
Volume of eluent (ml)	45	45	45
				Purity (%)	81.89	80.24	86.14
Yield (%)	66.7	74.3	71.6

From the table 7, it can be seen that under the condition of optimized sample dilution times, elution buffer flow rate and elution buffer ratio, the purity of the purified plasmid can reach about 80%, the yield can reach more than 66%, and the purpose of plasmid purification is achieved.

In addition, the purification method of the invention also greatly shortens the time required by the purification process. For example, for 200ml lysate, about 1.5 hours is required to complete the purification process using a conventional packed column such as plasmid select Xtra, while about 42 minutes is required using the method of the present invention (about 14 minutes for the first fixed column and about 28 minutes for the second fixed column), which is a half-time reduction.

It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method of purifying a plasmid comprising the steps of: (1) cracking the Escherichia coli culture containing the plasmid to be extracted to obtain a cracking solution; (2) carrying out chromatography by using a first fixed column to obtain a crude purified solution; (3) and (5) carrying out chromatography on the crude purification solution by using a second fixed column to obtain purified plasmid.

2. The method of claim 1, further comprising: after the step (1), the lysate is clarified to obtain a lysis supernatant, and the lysis supernatant is subjected to chromatography by using a first fixed column.

3. The method of claim 1, wherein the first fixed columns are DEAE fixed columns.

4. The method of claim 1, wherein the second fixed posts are C4 hydrophobic fixed posts.

5. The method of claim 1 wherein the first fixed column has a sample conductivity of 35 to 45 mS/cm.

6. The method of claim 1, wherein the first fixation column employs a first equilibration buffer comprising 5-15mM EDTA and 10-75mM Tris (ph 7.2).

7. The method of claim 1, wherein the second equilibration buffer employed with the second fixation column comprises 2-4M ammonium sulfate, 5-15mM EDTA, and 10-75mM Tris (ph 7.2).

8. The method of claim 1, wherein the first fixed column employs a first elution buffer comprising 0.5-2M NaCl, 5-15mM EDTA, and 10-75mM Tris (ph 7.2).

9. The method of claim 1, wherein the second fixed column employs a second elution buffer comprising 5-15mM EDTA and 10-75mM Tris (ph 7.2).

10. The method of claim 1, wherein the first fixed column has a proportion of elution buffer of 80% -100%.

11. The method of claim 1, wherein the second fixed column has a proportion of elution buffer of 40% -60%.

12. The method of claim 1, wherein the first fixed column has an operating flow rate of 50 ml/min.

13. The method of claim 1, wherein the second fixed column has an operating flow rate of 8 to 50 ml/min.

14. The method of claim 1, wherein the second fixation column has a sample dilution factor of 1.