阅读说明：本技术 一种慢病毒包装系统辅助质粒的超螺旋结构纯度的检测方法 (Detection method for supercoiled structure purity of helper plasmid of lentivirus packaging system ) 是由 张同存 张琴星 马传艳 彭波 于 2020-11-16 设计创作，主要内容包括：本发明涉及高效液相色谱法检测一种慢病毒包装系统辅助质粒超螺旋含量的方法,利用高效液相色谱法进行测定,通过回收高效液相色谱主峰,并且进行鉴定,其可以作为质粒超螺旋结构工作标准品。结果表明,用该方法对BZK2质粒超螺旋结构进行检测,其纯度达84.71％,该方法专属性强、操作简单、灵敏度高、结果准确,适用于质粒制品超螺旋结构纯度的检测。(The invention relates to a method for detecting the supercoiled content of helper plasmid of a lentivirus packaging system by high performance liquid chromatography, which utilizes the high performance liquid chromatography to carry out determination and can be used as a working standard substance of the supercoiled structure of the plasmid by recovering and identifying the main peak of the high performance liquid chromatography. The result shows that the purity of the BZK2 plasmid supercoiled structure detected by the method reaches 84.71 percent, and the method has strong specificity, simple operation, high sensitivity and accurate result, and is suitable for detecting the purity of the supercoiled structure of the plasmid product.)

1. A high performance liquid chromatography detection method for supercoiled purity of plasmid comprises the following steps:

(1) using a Waters e2695 model high performance liquid chromatograph and a Secre Proteomix SAX strong anion exchange chromatography column;

(2) and (3) chromatographic detection conditions:

firstly, the detection wavelength is 260 nm;

② the flow rate is 0.5 mL/min;

the temperature of the column is 30-35 ℃;

fourthly, the sample loading quantity is 20 mu L;

fifthly, the mobile phase A is 20mmol/LTris solution with the pH value of 7.5-8.0;

sixthly, the mobile phase B is a solution with the pH value of 7.5-8.0 of NaClpHs of 20mmol/L of LTris 1 mol/L;

seventhly, the elution gradient changes by 1 percent;

(3) preparation of working standard of each plasmid configuration: obtaining a linear structure working standard product by adopting a single enzyme digestion mode; obtaining working standard products of an open-loop structure and a super-spiral structure by adopting a mode of recovering a foreign peak and a main peak in a liquid chromatogram, and verifying and confirming the obtained working standard products of the undetermined open-loop structure and the super-spiral structure again by combining an agarose gel electrophoresis method and a high performance liquid chromatography method;

(4) and (3) detecting the purity of the supercoiled structure of the sample by using the confirmed supercoiled structure working standard substance in the step (3) and combining the chromatographic detection conditions in the step (2).

2. The method of claim 1, wherein the column temperature in step (2) is 35 ℃ and the mobile phase pH is 8.0.

3. The method according to claim 1, wherein the step (3) comprises in particular the steps of:

(1) preparation of working standard substance with plasmid linear structure

Taking 2-5 mu g of plasmid, carrying out single enzyme digestion on the plasmid by MluI-HF, carrying out alcoholization on a single enzyme digestion product, and dissolving the alcoholized product to 50 mu L by using a mobile phase A, thus obtaining a BZ2K plasmid linear plasmid working standard solution;

(2) preparation of working standard substance with plasmid open-loop structure

Injecting a sample of the plasmid stock solution by using the chromatographic detection condition in the step (2) of claim 1, recovering a peak of a mixed peak about 1 minute before a main peak in a chromatogram, alcoholizing the recovered plasmid DNA, and dissolving a purified product to 50 μ L by using a mobile phase A to obtain a plasmid open-loop structure working standard product;

(3) preparation of working standard substance with plasmid supercoiled structure

The chromatographic detection condition in the step (2) of claim 1 is utilized, the BZ2K plasmid stock solution is injected, the main peak in a chromatogram is recovered, the recovered plasmid DNA is alcoholized, and the product after alcoholization is dissolved to 100 μ L by using the mobile phase A.

4. The method according to claim 1, wherein the step (4) comprises in particular the steps of:

(1) blank non-interfering experiment: putting 100 mu L of the mobile phase A solution into a sample tray, and continuously sampling for 2 times, wherein 20 mu L of the solution is obtained each time;

(2) experiment of system applicability: putting 200 μ L of the system applicability solution into a sample tray, continuously feeding samples for 6 times according to the detection conditions of the step (2) of claim 1, feeding 20 μ L of each sample, recording chromatogram, taking out a system applicability report, and calculating the separation degree of main peak, theoretical plate number, tailing and RSD

(3) Drawing a standard curve: placing each standard substance described in claim 3 into a sample tray, sequentially injecting samples according to a well-established chromatographic condition, injecting samples for 2 times for each sample, injecting samples for 20 μ L each time, and drawing a standard curve and a linear regression equation by using the peak area and the concentration of supercoiled plasmids;

(4) and (3) testing a test solution: putting 100 mu L of sample solution into a sample tray, carrying out sample injection according to a well-formulated chromatographic condition, carrying out sample injection for each sample for 2 times, carrying out sample injection for 20 mu L each time, and recording a chromatogram;

(5) the calculation method comprises the following steps: purity C ═ Cx/C_MeasuringX 100%. Cx is the concentration of the supercoiled plasmid of the test sample; c_MeasuringThe plasmid concentration is determined by ultramicro UV spectrophotometry.

Technical Field

The invention relates to the field of pharmaceutical analysis, in particular to a method for detecting the purity of a plasmid DNA supercoiled structure by using a high performance liquid chromatography.

Background

Plasmids (plasmids) are widely present in the kingdom of life, ranging from bacteria, actinomycetes, filamentous fungi, macrofungi, yeasts to plants and even in the human body. From the viewpoint of molecular composition, there are DNA plasmids and also RNA plasmids; from the viewpoint of molecular configuration, there are linear plasmids and circular plasmids, and their phenotypes are also various. Bacterial plasmids are the most commonly used vectors in genetic engineering. Plasmid (plasmid) is a genetic component outside bacterial or cellular chromatin, capable of autonomous replication, symbiotic with bacteria or cells, is double-stranded covalently closed circular DNA outside chromatin, and can naturally form a supercoiled structure.

Plasmid DNA is a circular double-stranded DNA molecule capable of autonomous replication outside bacterial chromosomes. The molecular weights of different types of plasmid DNA are different and can reach 10⁵kDa, small is only 10³kDa. Plasmid DNA has three different configurations:

covalently closed circular DNA exhibits a supercoiled structure (SC configuration) which, in the electropherogram, is in front of the gel;

one strand maintains a complete ring structure, and the other strand has one to a plurality of gaps and ring-opened DNA which presents a ring-opened configuration (OC configuration) and walks behind the gel;

③ the double bond-broken DNA presents a linear structure (L configuration) which runs in the middle of the gel.

Because the transfection efficiency of the supercoiled plasmid DNA is high, the dosage of the drug can be greatly reduced, so that the plasmid DNA used in DNA vaccines, gene therapy and cell therapy is in a supercoiled configuration. But the supercoiled structure of the plasmid DNA can be damaged in the process of plasmid purification, and plasmid DNA with various configurations can be formed, so that the plasmid packaging efficiency is influenced, and therefore, accurate quantitative analysis on the supercoiled content of the plasmid DNA is necessary.

The existing technology for detecting the content of the supercoiled DNA comprises an agarose gel electrophoresis method and a capillary gel electrophoresis method, but the existing technology has the defects of the detection method, for example, the capillary gel electrophoresis method needs a complicated sample pretreatment process and is not suitable for being used in each step of plasmid DNA production; agarose gel electrophoresis at OD₂₆₀The detection range at the wavelength is too narrow, and errors are easily caused in the sample dilution process, the accuracy is low, and the method is not suitable for each step of plasmid DNA production.More importantly, neither of the above methods quantitatively detects the content of supercoiled DNA.

Disclosure of Invention

Aiming at the defects and shortcomings in the prior art, the invention aims to provide a method for detecting the purity of a plasmid supercoiled structure by high performance liquid chromatography, wherein a Waters e2695 type high performance liquid chromatograph is used for performing high performance liquid chromatography analysis and detection on a lentivirus packaging system auxiliary plasmid BZ2K, and the proportion of the supercoiled structure in a sample can be rapidly calculated by adopting an external standard method.

In one aspect of the invention, a high performance liquid chromatography detection method for supercoiled purity of plasmid is provided, which comprises the following steps:

(1) using a Waters e2695 model high performance liquid chromatograph and a Secre Proteomix SAX strong anion exchange chromatography column

(2) Selection of detection conditions:

firstly, the detection wavelength is 260 nm;

② the flow rate is 0.5 mL/min;

the temperature of the column is 35 ℃;

fourthly, the sample loading quantity is 20 mu L;

mobile phase A is 20mmol/L Tris pH8.0 solution;

sixthly, the mobile phase B is 20mmol/L Tris 1mol/L NaCl pH8.0 solution;

seventhly, the change amount of the elution gradient is 1 percent.

(3) Preparation of working standard of each plasmid configuration: obtaining a linear structure working standard product by adopting a single enzyme digestion mode; obtaining working standard products of an open-loop structure and a super-spiral structure by adopting a mode of recovering a foreign peak and a main peak in a liquid chromatogram, and verifying and confirming the obtained working standard products of the undetermined open-loop structure and the super-spiral structure again by combining an agarose gel electrophoresis method and a high performance liquid chromatography method;

(4) and (3) detecting the purity of the supercoiled structure of the sample by using the confirmed supercoiled structure working standard substance and combining the optimized chromatographic detection conditions.

In summary, the beneficial effects of the invention at least include the following:

1. obtaining a set of chromatographic purification parameters by groping, wherein the purity of the supercoiled structure plasmid obtained under the set of chromatographic purification parameters can reach more than 99 percent;

2. the method is simple, convenient, fast and high in sensitivity, and has higher resolution than the conventional gel electrophoresis method.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a diagram of the structure of the BZ2K plasmid;

FIG. 2 is a TSKgel DNA-NPR column BZ2K plasmid high performance liquid chromatogram;

FIG. 3 high performance liquid chromatogram of BZ2K plasmid with an elution gradient changed by 0.8% in an optimization experiment;

FIG. 4 shows a high performance liquid chromatogram of the BZ2K plasmid when the elution gradient was changed by 1% in an optimization experiment;

FIG. 5 is a high performance liquid chromatogram of BZ2K under the preferred parameters of the plasmid;

FIG. 6 agarose gel electrophoresis of each configuration of plasmid;

FIG. 7 BZ2K plasmid supercoiled standard chromatogram;

FIG. 8 BZ2K plasmid is added with open circular structure chromatogram;

FIG. 9 BZ2K plasmid with supercoiled, linear structure chromatogram;

FIG. 10 shows the results of gel electrophoresis for detecting the purity of the working standard substance of the supercoiled structure of the plasmid BZ 2K;

FIG. 11 blank sample chromatogram;

FIG. 12 System suitability verification results;

FIG. 13 standard curve.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: screening and optimization of assay conditions

The BZ2K plasmid is one of the essential helper plasmids in the packaging process of lentiviruses, and the structure of the plasmid is shown in FIG. 1. The respective structures of BZ2K plasmid were separated using a Waters e2695 type high performance liquid chromatograph and a TSKgel DNA-NPR column.

Experiment 1:

the detection wavelength is 260 nm;

the flow rate is 0.5 ml/min;

the column temperature is 35 ℃;

loading 20 μ L (2 μ g);

mobile phase A: 20mmol/L Tris pH8.8 solution;

mobile phase B: 20mmol/L Tris solution with 1mol/L NaCl pH8.8;

elution gradient 1% (table 1).

TABLE 1

Time (minutes)	Mobile phase A%	Mobile phase B%
			0	50	50
5	50	50
			45	10	90
50	50	50

As shown in FIG. 2, it was found that the separation effect of different components (supercoiled plasmid and open-loop plasmid) of BZ2K plasmid was not sufficient in the chromatogram of TSKgel DNA-NPR column, and the degree of separation was only 1.9. Based on the poor effect, the other chromatographic column Proteomix SAX strong anion exchange chromatography is replaced, and the pH value, the column temperature and the elution gradient of the mobile phase are optimized and screened.

Experiment 2: screening for mobile phase pH

Adjusting the pH values of the mobile phases A and B to four groups of 7.5, 8.0, 8.5 and 9.0; the rest of the conditions were the same as in experiment 1, and the results were as follows:

TABLE 2

The results show that the pH of mobile phases a and B is preferably 8.0.

Experiment 3: screening of column temperature

Adjusting column temperature to three groups of 25 deg.C, 30 deg.C, 35 deg.C and 40 deg.C; the rest of the conditions were the same as in experiment 1, and the results were as follows:

TABLE 3

Column temperature grouping	Degree of separation of main peak	USP tailing	Number of theoretical plates of USP
				25℃	1.780600e+000	1.311389e+000	1.238183e+004
30℃	1.820771e+001	1.106584e+000	3.235684e+003
				35℃	1.951852e+000	1.021085e+000	3.951557e+003
40℃	1.965713e+000	1.251282e+001	2.542138e+004

The results show that column temperature adjustment to 30-35 ℃ is preferred.

Experiment 4: screening for elution gradient

The elution gradient was set to 0.8%, 1.0% in two groups, and the rest of the conditions were the same as in experiment 1. The results are shown in FIGS. 3-4, where the peak pattern appears more severely advanced (main peak tailing factor of 0.85) when the elution gradient change is 0.8%, suggesting that an elution gradient of 1.0% is better than an elution gradient of 0.8%.

Experiment 5: determining optimal detection conditions

Through a series of single factor experiments, the optimal detection conditions for distinguishing supercoiled plasmids from open-loop structure plasmids are determined as follows: the detection wavelength is 260 nm; the flow rate is 0.5 ml/min; the column temperature is 30-35 ℃; the sample loading amount is 20 mu L; the mobile phase A is 20mmol/L Tris pH8.0 solution; the mobile phase B is 20mmol/L Tris 1mol/L NaCl pH8.0 solution; the gradient elution was changed by 1%, as shown in table 4:

TABLE 4

Time (minutes)	Mobile phase A%	Mobile phase B%
			0	50	50
5	25	75
			25	5	95
30	50	50

Under the detection condition, the map of the BZ2K plasmid is shown in FIG. 5, and the main component peak and the small peak of the BZ2K plasmid can be clearly seen, wherein the main component peak is a supercoiled plasmid, and the small peak is an open-loop plasmid.

Example 2: preparation of work standard substance of BZ2K plasmid in each configuration

1. Preparation of BZ2K plasmid linear structure working standard

Taking 2 mu g-5 mu g of BZ2K plasmid, carrying out single enzyme digestion by MluI-HF, carrying out alcoholization on the single enzyme digestion product, and dissolving the alcoholized product to 50 mu L by using a mobile phase A, thus obtaining the BZ2K plasmid linear plasmid working standard solution.

2. Preparation of BZ2K plasmid open-loop structure work standard

The optimal chromatographic detection conditions (the detection wavelength is 260nm, the flow rate is 0.5ml/min, the column temperature is 35 ℃, the sample loading amount is 20 mu L, the mobile phase A is 20mmol/L Tris pH8.0 solution, the mobile phase B is 20mmol/L Tris 1mol/L NaCl pH8.0 solution, the gradient elution is 1 percent) determined in the example 1 are utilized, the BZ2K plasmid stock solution is injected, the peak recovery is carried out on the hybrid peak (the hybrid peak about 1 minute before the main peak) in the chromatogram, the recovered plasmid DNA is alcoholized, and the purified product is dissolved to 50 mu L by the mobile phase A, namely the BZ2K plasmid open-loop structure work standard product.

3. Preparation of BZ2K plasmid supercoiled structure work standard

Sample introduction is carried out on BZ2K plasmid stock solution by utilizing the finally determined chromatographic detection conditions in the example 1, the main peak in a chromatogram is recovered, the recovered plasmid DNA is alcoholized, and the product after alcoholization is dissolved to 100 mu L by using a mobile phase A.

The recovered hetero-peak (open-loop structure) and main peak (supercoiled structure) were re-identified using two methods:

(1) agarose gel electrophoresis:

as shown in FIG. 6, the electrophoretic bands of the single digestion product, the recovered hetero-peak (open-loop structure) and the main peak (supercoiled structure) are clearly positioned and correspond to the electrophoretic bands of the plasmid stock solution one by one.

(2) HLPC method:

the recovered main component peak was again loaded according to the chromatographic conditions found in example 1, as shown in FIG. 7, there were a main peak (18.911min) and a hetero-peak (17.918min), which is only a few thousandths of the main peak; the recovered hetero-peak (about 1 minute before the main peak) was added to the recovered main component peak, and as shown in fig. 8, a significant peak increase occurred at the hetero-peak position (17.862min) in the original chromatogram; the single digestion product of BZ2K plasmid was added to the recovered main component peak, and as shown in FIG. 9, a clear chromatographic peak (18.031min) appeared between the main peak and the hetero peak.

The purity of the BZ2K plasmid supercoiled structure working standard is detected by agarose gel electrophoresis, as shown in figure 10, 200ng of HPLC peak (main peak) recovery solution is taken for agarose gel electrophoresis, the purity of the main band (supercoiled) is calculated to be 99.23 percent and is more than 99.0 percent of the minimum peak purity (when the peak separation degree is 1.5, the peak purity is 99.0 percent), namely the main peak of a chromatogram is a single substance, and the chromatographic condition for detection can effectively separate the BZ2K plasmid supercoiled structure from other BZ2K plasmid components (open-loop and linear structure plasmids). Therefore, both methods can prove that the chromatographic conditions obtained in example 1 obtain the supercoiled structure with high purity, and the supercoiled structure can be used as a working standard of the supercoiled structure of the BZ2K plasmid.

Example 3: system applicability solution preparation

Dissolving 50 mu L of linear plasmid working standard substance, 50 mu L of open-loop plasmid working standard substance and 20 mu g of supercoiled plasmid working standard substance into 200 mu L of mobile phase A, and mixing uniformly to obtain the system applicability solution.

Example 4: preparation of standard curve solution

Taking BZ2K plasmid working standard products with different volumes according to the concentration of BZ2K plasmid working standard product solution and the purity of supercoiled plasmid, respectively diluting the working standard products with a mobile phase A into 100 mu L solutions with supercoiled plasmid concentrations of 50 ng/mu L, 100 ng/mu L, 150 ng/mu L, 200 ng/mu L and 250 ng/mu L, and uniformly mixing to obtain the supercoiled plasmid.

Example 5: test solution treatment

And (3) according to the DNA concentration of the BZ2K plasmid product obtained by the test, diluting the PBZ2K plasmid product into 200 muL of solution with the concentration of 100 ng/muL by using the mobile phase A, and obtaining the test solution.

Example 6: detection method

1. Blank non-interfering experiment: and (3) putting 100 mu L of the mobile phase A solution into a sample tray, carrying out continuous sample injection for 2 times, wherein 20 mu L of the solution is injected each time, and recording a chromatogram map. As shown in FIG. 11, no chromatographic peak was observed in the chromatogram from 4min to 30 min.

2. Experiment of system applicability: 200 mu L of the system applicability solution is put into a sample tray, and according to the well-established chromatographic conditions (the detection wavelength is 260nm, the flow rate is 0.5ml/min, the column temperature is 35 ℃, the sample loading amount is 20 mu L, the mobile phase A is 20mmol/L Tris pH8.0 solution, the mobile phase B is 20mmol/L Tris 1mol/L NaCl pH8.0 solution, the gradient elution is 1 percent), the continuous sample introduction is carried out for 6 times, 20 mu L of sample introduction is carried out each time, the chromatogram is recorded, the system applicability report is taken out, and the main peak separation degree, the theoretical plate number, the tailing and the RSD (peak area and peak output time) are calculated (Table 4). As shown in FIG. 12, the main peak was completely separated from the impurity peak, and the peak heights of the open-loop structure and linear structure of the plasmid reached about one tenth of the peak height of the main peak.

TABLE 4 separation of the applicable solutions of the System

3. Drawing a standard curve (external standard curve method): the standard curve solution was placed in a sample tray and samples were sequentially injected according to the established chromatographic conditions, 2 samples were injected for each time, 20 μ L for each sample, and the standard curve and the linear regression equation were plotted using the peak area (average of two times) and the concentration of supercoiled plasmid, as shown in fig. 13.

4. And (3) testing a test solution: and (3) putting 100 mu L of sample solution into a sample tray, injecting samples according to the well-formulated chromatographic conditions, injecting samples for each sample for 2 times, injecting samples for 20 mu L each time, and recording a chromatogram.

5. The calculation method comprises the following steps: purity C ═ Cx/C_MeasuringX 100%. Cx is the concentration of the supercoiled plasmid of the test sample; c_MeasuringThe plasmid concentration was determined by ultraminiUV spectrophotometry (Q5000).

Example 7: results and analysis of the experiments

Through chromatogram and experimental data analysis (table 5-table 6), under the condition of the detection method, the sample concentration of BZ2K plasmid supercoiled structure plasmid is between 49.94 ng/muL and 249.71 ng/muL, the supercoiled plasmid in the plasmid is completely and effectively separated from other two structure plasmids, the concentration of the supercoiled plasmid can be rapidly and accurately calculated through an external standard method, and the purity of the supercoiled plasmid is calculated to be 84.71%.

TABLE 5

TABLE 6

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.