阅读说明：本技术 一种质粒dna原液制备方法 (Preparation method of plasmid DNA stock solution ) 是由 王雪云 郭土敬 吴彦萍 吴灼斌 梁富 黄致翔 叶才文 刘爱和 曹静 梁琳琳 郭采 于 2021-08-12 设计创作，主要内容包括：本发明公开了一种质粒DNA原液制备方法,其包括以下步骤：平衡质粒DNA液温度至常温,进行预浓缩至质粒DNA终浓度为500～1000μg/ml,得预浓缩液；采用含有多元醇的洗滤缓冲液对预浓缩液进行等体积洗滤,再浓缩至合适浓度。本发明的质粒DNA原液制备方法通过控制温度条件,并同时采用质粒DNA低浓度预浓缩及通过含多元醇的洗滤缓冲液进行洗滤,可有效减少质粒DNA的聚集沉淀及机械损伤,显著提高了质粒DNA的超滤回收率,回收率可高达85％以上。(The invention discloses a preparation method of plasmid DNA stock solution, which comprises the following steps: balancing the temperature of the plasmid DNA liquid to normal temperature, and carrying out preconcentration until the final concentration of the plasmid DNA is 500-1000 mug/ml, so as to obtain a preconcentration liquid; and (3) washing and filtering the pre-concentrated solution by adopting a washing and filtering buffer solution containing polyol in an equal volume, and then concentrating to a proper concentration. The preparation method of the plasmid DNA stock solution can effectively reduce the aggregation precipitation and the mechanical damage of the plasmid DNA by controlling the temperature condition, adopting the low-concentration pre-concentration of the plasmid DNA and carrying out the washing and filtering by the washing and filtering buffer solution containing the polyalcohol at the same time, obviously improves the ultrafiltration recovery rate of the plasmid DNA, and the recovery rate can reach more than 85 percent.)

1. A method for preparing a plasmid DNA stock solution is characterized by comprising the following steps: balancing the temperature of the plasmid DNA liquid to normal temperature, and carrying out preconcentration until the final concentration of the plasmid DNA is 500-1000 mug/ml, so as to obtain a preconcentration liquid; and (3) washing and filtering the pre-concentrated solution by adopting a washing and filtering buffer solution containing polyol in an equal volume, and then concentrating to a proper concentration.

2. The method for preparing a plasmid DNA stock solution according to claim 1, which specifically comprises the steps of:

(1) temperature balance: balancing the temperature of the plasmid DNA liquid to normal temperature, wherein the normal temperature is 20-30 ℃;

(2) pre-concentration: carrying out pre-concentration on the balanced plasmid DNA by adopting ultrafiltration, concentrating until the final concentration of the plasmid DNA is 500-1000 mug/ml, and maintaining the same volume until the pre-concentration is finished to obtain a pre-concentrated solution;

(3) washing and filtering: washing and filtering the pre-concentrated solution by using a washing and filtering buffer solution containing polyalcohol in an equal volume;

(4) concentration: after the washing and filtering are finished, the pre-concentrated solution is concentrated to a proper concentration.

3. The method for preparing a plasmid DNA stock solution according to claim 1 or 2, wherein the polyol is sorbitol, mannitol, pentaerythritol, glycerol or xylitol.

4. The method for preparing a plasmid DNA stock solution according to claim 1 or 2, wherein the mass concentration of the polyol is 1 to 10% (W/V).

5. The method for preparing a plasmid DNA stock solution according to claim 1 or 2, wherein in the step (1), the plasmid DNA is purified plasmid DNA.

6. The method for preparing a plasmid DNA stock solution according to claim 1 or 2, wherein in the step (2), the ultrafiltration device for ultrafiltration is a membrane cartridge or a hollow fiber column.

7. The method for preparing a plasmid DNA stock solution according to claim 6, wherein the pore size of the membrane or hollow fiber column is 100 to 500 kD.

8. The method for preparing a plasmid DNA stock solution according to claim 1 or 2, wherein sodium chloride is not added to the washing buffer solution in the step (3).

9. The method for preparing the plasmid DNA stock solution according to claim 1 or 2, wherein the washing filtration ratio of the medium-volume washing filtration in the step (3) is 3 to 10 times.

10. The method for preparing a plasmid DNA stock solution according to claim 1 or 2, wherein the appropriate concentration is 2.3 to 3.0 mg/ml.

Technical Field

The invention relates to the field of biological pharmacy, and particularly relates to a preparation method of a plasmid DNA stock solution.

Background

After purification, the plasmid DNA is usually concentrated to a pharmaceutical quality standard. But because plasmid DNA during concentration, with the concentration increase, its viscosity also increases significantly. This not only easily causes damage to the supercoiled structure of plasmid DNA, but also seriously affects the recovery rate of plasmid DNA, resulting in an increase in pharmaceutical cost. In the prior art, hollow fibers or membranes are usually adopted to concentrate purified plasmid DNA, and PBS buffer solution is adopted for washing and filtering, but the plasmid recovery rate is usually maintained between 60% and 70%, and the plasmid supercoiled configuration in ultrafiltration concentration often has mechanical damage, which causes the reduction of plasmid quality.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a preparation method of a plasmid DNA stock solution.

The technical problem to be solved by the invention is realized by the following technical scheme:

a method for preparing a plasmid DNA stock solution comprises the following steps: balancing the temperature of the plasmid DNA liquid to normal temperature, and carrying out preconcentration until the final concentration of the plasmid DNA is 500-1000 mug/ml, so as to obtain a preconcentration liquid; and (3) washing and filtering the pre-concentrated solution by adopting a washing and filtering buffer solution containing polyol in an equal volume, and then concentrating to a proper concentration.

As a preferred embodiment of the method for preparing the plasmid DNA stock solution, the method specifically comprises the following steps:

(1) temperature balance: balancing the temperature of the plasmid DNA liquid to normal temperature, wherein the normal temperature is 20-30 ℃;

(2) pre-concentration: carrying out pre-concentration on the balanced plasmid DNA by adopting ultrafiltration, concentrating until the final concentration of the plasmid DNA is 500-1000 mug/ml, and maintaining the same volume until the pre-concentration is finished to obtain a pre-concentrated solution;

(3) washing and filtering: washing and filtering the pre-concentrated solution by using a washing and filtering buffer solution containing polyalcohol in an equal volume;

(4) concentration: after the washing and filtering are finished, the pre-concentrated solution is concentrated to a proper concentration.

As a preferred embodiment of the method for preparing the plasmid DNA stock solution provided by the invention, the polyhydric alcohol is sorbitol, mannitol, pentaerythritol, glycerol or xylitol.

As a preferred embodiment of the method for preparing the plasmid DNA stock solution provided by the invention, the mass concentration of the polyhydric alcohol is 1-10% (W/V).

As a preferred embodiment of the method for preparing the plasmid DNA stock solution provided by the invention, in the step (1), the plasmid DNA is purified plasmid DNA.

In a preferred embodiment of the method for preparing a plasmid DNA stock solution according to the present invention, in the step (2), the ultrafiltration device for ultrafiltration is a membrane module or a hollow fiber column.

As a preferred embodiment of the preparation method of the plasmid DNA stock solution provided by the invention, the pore diameter of the membrane package or the hollow fiber column is 100-500 kD.

As a preferred embodiment of the preparation method of the plasmid DNA stock solution provided by the invention, the phosphate concentration in the washing and filtering buffer solution in the step (3) is 10-100 mmol/L.

As a preferred embodiment of the method for preparing the plasmid DNA stock solution provided by the invention, sodium chloride is not added to the washing buffer solution in the step (3).

As a preferred embodiment of the preparation method of the plasmid DNA stock solution provided by the invention, the washing and filtering times of the medium-volume washing and filtering in the step (3) are 3-10 times.

As a preferred embodiment of the preparation method of the plasmid DNA stock solution provided by the invention, the proper concentration is 2.3-3.0 mg/ml.

The invention has the following beneficial effects:

the preparation method of the plasmid DNA stock solution can effectively reduce the aggregation precipitation and the mechanical damage of the plasmid DNA by controlling the temperature condition, simultaneously adopting the low-concentration pre-concentration of the plasmid DNA and carrying out the washing and filtering by the washing and filtering buffer solution containing the polyalcohol, the supercoiled content can reach more than 93 percent, the ultrafiltration recovery rate of the plasmid DNA is obviously improved, and the recovery rate can reach more than 85 percent. The invention does not need to change the whole ultrafiltration process, can obviously increase the ultrafiltration concentration recovery rate of the plasmid DNA only by processing the concentration and the temperature of the plasmid DNA and the formula of the washing and filtering buffer solution, has simple operation and high repeatability, and is easy for industrial large-scale application.

Drawings

FIG. 1 is a schematic process flow diagram of the present invention;

FIG. 2 is a diagram of agarose gel electrophoresis results of an embodiment of the invention, wherein lanes 1 to 5 correspond to the agarose gel electrophoresis results of the stock solutions of groups 1 to 5, respectively;

FIG. 3 is a diagram of the results of the agarose gel electrophoresis of the second stock solution of the present invention, wherein lanes 1 to 5 correspond to the agarose gel electrophoresis of the first stock solution of group 1 to group 5, respectively;

FIG. 4 is a diagram of the results of three-run agarose gel electrophoresis in accordance with the present invention, wherein lanes 1 to 5 correspond to run-on agarose gel electrophoresis results of groups 1 to 5, respectively;

FIG. 5 is a diagram of the results of four bulk agarose gel electrophoresis in accordance with the present invention, wherein lanes 1 to 5 correspond to the results of the four bulk agarose gel electrophoresis of groups 1 to 5, respectively;

FIG. 6 is a graph showing the results of the agarose gel electrophoresis of comparative example 1, in which lane 1 shows the results of the agarose gel electrophoresis of comparative example 1.

Detailed Description

As described in the background art, in the prior art, hollow fibers or membrane membranes are usually used to concentrate the purified plasmid DNA, and PBS buffer solution is used for washing and filtering, but the plasmid recovery rate is usually maintained between 60% and 70%, and the plasmid supercoiled configuration in the ultrafiltration concentration often has mechanical damage, which causes the reduction of plasmid quality.

In order to solve the technical problems, the invention provides a preparation method of a plasmid DNA stock solution, which comprises the following steps: balancing the temperature of the plasmid DNA liquid to normal temperature, and carrying out preconcentration until the final concentration of the plasmid DNA is 500-1000 mug/ml, so as to obtain a preconcentration liquid; and (3) washing and filtering the pre-concentrated solution by adopting a washing and filtering buffer solution containing polyol in an equal volume, and then concentrating to a proper concentration.

The preparation method of the plasmid DNA stock solution can effectively reduce the aggregation precipitation and the mechanical damage of the plasmid DNA by controlling the temperature condition, simultaneously adopting the low-concentration pre-concentration of the plasmid DNA and carrying out the washing and filtering by the washing and filtering buffer solution containing the polyalcohol, the supercoiled content can reach more than 93 percent, the ultrafiltration recovery rate of the plasmid DNA is obviously improved, and the recovery rate can reach more than 85 percent.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The invention will be further illustrated by the following examples. The ultrafiltration membrane package and the fixture in the method of the invention are both Mercury. The reagents used were all pharmaceutical grade.

The recovery rate calculation method comprises the following steps: the recovery rate (volume of the DNA vaccine stock solution x concentration of the DNA vaccine stock solution)/(volume of the DNA purified solution x concentration of the DNA purified solution) x 100%

Example one

This example shows a method for preparing a plasmid DNA stock solution, which specifically comprises the following steps:

(1) temperature balance: the purified plasmid DNA solution is balanced to 25 ℃, and is divided into 5 parts after the concentration is determined;

(2) pre-concentration: concentrating the balanced purified plasmid DNA liquid with 300kD ultrafiltering membrane to final concentration of 500, 800, 1000, 1500 and 2000 μ g/ml, maintaining the same volume until the pre-concentration is completed to obtain pre-concentrated liquid;

(3) washing and filtering: washing and filtering the pre-concentrated solution by 5 times by using a washing and filtering buffer solution, wherein the concentration of mannitol in the washing and filtering buffer solution is 2.5%, the concentration of phosphate in the washing and filtering buffer solution is 50mmol/L, and the pH value of the washing and filtering buffer solution is 7.5;

(4) concentration: and (4) calculating the concentration value of the purified DNA solution to obtain the required concentration multiple of 2.5mg/ml, and concentrating according to the multiple.

The results of this example are shown in table 1, by changing the pre-concentration multiple, the plasmid concentration during pre-concentration is different, which causes the viscosity difference of the sample, when the pre-concentration plasmid concentration is 500-1000 μ g/ml, the plasmid recovery rate is above 85%, which reduces plasmid agglomeration and reduces supercoiled content loss caused by mechanical damage, and when the plasmid pre-concentration is increased to above 1500 μ g/ml, the viscosity is too high, part of the plasmid may be lost in the membrane package, which is difficult to elute and recover, resulting in the significant decrease of plasmid recovery rate.

Table 1 example data

Example two

This example shows a method for preparing a plasmid DNA stock solution, which specifically comprises the following steps:

(1) temperature balance: balancing the purified DNA solution to 25 ℃, and dividing the purified DNA solution into 5 parts after measuring the concentration;

(2) pre-concentration: respectively concentrating the balanced purified plasmid DNA liquid with a 300kD ultrafiltration membrane package until the final concentration is 1000 mug/ml, and maintaining the same volume until the preconcentration is finished to obtain a preconcentrated solution;

(3) washing and filtering: washing and filtering the pre-concentrated solution by 5 times by using a washing and filtering buffer solution, wherein the concentration of mannitol in the washing and filtering buffer solution is respectively 0%, 1%, 2.5%, 5% and 10%; the concentration of phosphate is 50mmol/L, and the pH value of the washing and filtering buffer solution is 7.5;

(4) concentration: and (4) calculating the concentration value of the purified DNA solution to obtain the required concentration multiple of 2.5mg/ml, and concentrating according to the multiple.

The results of this example are shown in Table 2, and no significant difference in plasmid recovery was found when the pre-concentrated plasmid concentration was 1000. mu.g/ml, regardless of whether mannitol was added or not, by washing the samples with varying mannitol concentration in the washing buffer. And the addition of 1-10% of mannitol can obviously reduce the loss of supercoiled content, which shows that mannitol can effectively reduce the oxidation or mechanical damage of plasmid DNA in the concentration process.

Table 2 example two data

EXAMPLE III

This example shows a method for preparing a plasmid DNA stock solution, which specifically comprises the following steps:

(1) temperature balance: dividing the purified DNA solution into four parts, and balancing to 4 ℃, 10 ℃, 20 ℃ and 30 ℃ respectively;

(2) pre-concentration: respectively concentrating the balanced purified plasmid DNA liquid with a 300kD ultrafiltration membrane package until the final concentration is 1000 mug/ml, and maintaining the same volume until the preconcentration is finished to obtain a preconcentrated solution;

(3) washing and filtering: washing and filtering the pre-concentrated solution by 5 times by using a washing and filtering buffer solution, wherein the concentration of mannitol in the washing and filtering buffer solution is 2.5 percent, the concentration of phosphate is 50mmol/L, and the pH value of the washing and filtering buffer solution is 7.5;

(4) concentration: and (4) calculating the concentration value of the purified DNA solution to obtain the required concentration multiple of 2.5mg/ml, and concentrating according to the multiple.

The results of this example are shown in table 3, and it was found that the recovery of plasmid was significantly lower than 20-37 ℃ below 20 ℃ by ultrafiltration concentration at different temperatures. And the plasmid recovery rate and the supercoiled content of the treatment group at 20-37 ℃ have no obvious difference. Wherein, the temperature of 37 ℃ is maintained in practical application, and the cost is higher, so the use of 20-30 ℃ is more suitable.

Table 3 example three data

Example four

This example shows a method for preparing a plasmid DNA stock solution, which specifically comprises the following steps:

(1) temperature balance: dividing the DNA purified solution into five parts after the concentration is measured, and balancing the temperature to 25 ℃;

(2) pre-concentration: respectively concentrating the balanced purified plasmid DNA liquid with a 300kD ultrafiltration membrane package until the final concentration is 1000 mug/ml, and maintaining the same volume until the preconcentration is finished to obtain a preconcentrated solution;

(3) washing and filtering: washing and filtering the pre-concentrated solution by 5 times by using a washing and filtering buffer solution, wherein the concentration of polyhydric alcohols in the washing and filtering buffer solution is respectively 2.5%, the concentration of phosphate is 50mmol/L, and the pH value of the washing and filtering buffer solution is 7.5, wherein the polyhydric alcohols are respectively mannitol, glycerol, sorbitol and xylitol;

(4) concentration: and (4) calculating the concentration value of the purified DNA solution to obtain the required concentration multiple of 2.5mg/ml, and concentrating according to the multiple.

The results of this example are shown in Table 4, and by performing the ultrafiltration using different polyols, it is shown that whether the addition of polyol does not affect the concentration recovery, but has a significant effect on the supercoiled content. And the ultrafiltration concentration recovery rate and the supercoiled content of different types of polyols have no obvious difference. Therefore, in the ultrafiltration concentration washing and filtering process, the polyalcohol added into the washing and filtering buffer solution can effectively reduce the oxidative damage or mechanical damage of plasmids and improve the product quality.

Table 4 example four data

Comparative example 1

This example shows a method for preparing a plasmid DNA stock solution, which specifically comprises the following steps:

(1) temperature balance: the purified plasmid DNA solution is balanced to 2-8 ℃; the plasmid DNA sample before ultrafiltration is an intermediate product and is usually stored in a low-temperature environment;

(2) concentration: calculating concentration times required by concentration of the purified DNA solution to 2.5mg/ml, and concentrating according to the times;

(3) washing and filtering: and (3) washing and filtering the concentrated solution by 5 times by using a washing and filtering buffer solution, wherein the phosphate concentration in the washing and filtering buffer solution is 50mmol/L, the sodium chloride concentration is 150mmol/L, the pH value of the washing and filtering buffer solution is 7.5, and the washing and filtering buffer solution does not contain polyol.

Table 5 comparative example data

As can be seen from Table 5, the plasmid DNA stock solution is obtained by concentrating the purified plasmid DNA with hollow fibers or membranes and performing washing filtration with PBS buffer solution, but the damage and recovery rate of the supercoiled plasmid structure are generally low in ultrafiltration concentration. In general, in the prior art, 100-200mmol/L NaCl is added to the washing buffer to facilitate the plasmid DNA dissolution and to facilitate the plasmid recovery. However, the inventor of the present invention conducted the existing stock solution preparation process to find out unintentionally, after adding 100mmol/L sodium chloride conventionally, the plasmid dissolution will be reduced to affect the recovery rate of plasmid, wherein the longer the plasmid length, the more sensitive the sodium chloride concentration, i.e. the plasmid DNA is extremely easy to be partially precipitated in the membrane package or hollow fiber column during the concentration and washing filtration process to seriously affect the recovery rate. The invention further provides a preparation method of the plasmid DNA stock solution by carrying out a large number of experiments, and particularly, the plasmid DNA before concentration is balanced to be in a normal temperature state by controlling the temperature condition, so that the problems of plasmid agglomeration, mechanical damage and the like in the ultrafiltration process due to the obvious increase of the viscosity of the plasmid DNA under the low-temperature condition in the prior art are solved; the invention also adopts low-concentration pre-concentration of plasmid DNA, thus solving the problems that the high concentration of DNA causes the viscosity of the DNA to be increased and the plasmid agglomeration, mechanical damage and the like are more easily caused in the prior art; the invention further carries out washing filtration by the washing filtration buffer solution containing the polyalcohol, thereby solving the problem that the plasmid quality is reduced due to the lack of protection on the pH value, the oxidability and the mechanical damage of the plasmid DNA in the washing filtration operation process after concentration in the prior art; in addition, the used washing and filtering buffer solution does not contain sodium chloride, so that the influence on the dissolution of plasmid DNA after the sodium chloride is added is avoided.

It should be noted that, as can be seen from tables 1 to 3 and 5, the technical effect of the present invention is the sum of the synergistic effects of the technical features of the respective steps, and the steps have certain inherent correlation, and are not the simple superposition of the effects of the individual technical features. The present invention can reduce the aggregation and precipitation and mechanical damage of plasmid DNA effectively, raise the ultrafiltering recovery rate of plasmid DNA to over 93%, and raise the recovery rate up to over 85%. The above effects produced by the present invention are obtained by the mutual synergy, and are inseparable. In addition, the whole ultrafiltration process is not required to be changed, the ultrafiltration concentration recovery rate of the plasmid DNA can be obviously increased only by processing the concentration of the plasmid DNA, the temperature of the solution and the formula of the washing and filtering buffer solution, the operation is simple, the repeatability is high, and the industrial large-scale application is easy.

The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.