Title of Invention: Method for Purification of Nucleic Acids
technical field
[One]
The present application relates to a method for purifying a nucleic acid, and specifically, a first step of crystallizing the nucleic acid into a solution containing a hydrophilic organic solvent; And a second step of drying the crystallized nucleic acid with high-humidity hot air air; relates to a nucleic acid purification method comprising a.
background
[2]
A method using a hydrophilic organic solvent is mainly used as a method for crystallizing nucleic acids. In the case of nucleic acids used for food, the removal of the organic solvent in the final product is a very important problem due to the regulation of the amount of residual organic solvent by country. Hydrophilic organic solvents commonly used for crystallization of nucleic acids include ethanol and methanol, and in particular, complete removal of methanol is required because residual control is strict. Nevertheless, the method using an organic solvent (Korean Patent No. 10-0051324) is still generally used as a nucleic acid crystallization method. , Korean Patent No. 10-0025552, and Korean Patent No. 10-0117428, etc. also disclose a method of crystallizing a nucleic acid using an organic solvent.
[3]
Nucleic acid crystals, especially 5'-guanylate disodium heptahydrate crystals that exist in the form of heptahydrate, lose crystal water even at a low temperature of room temperature, and the amount varies from heptahydrate to 2.5hydrate when the temperature is raised from room temperature to 80°C for about 30 minutes. As a result, about 70% loss occurs (FIG. 1). The loss of hydrates in the 5'-disodium guanylate heptahydrate crystals decreases the crystallinity of the crystals, resulting in crystal weakening and crystalline change, so it is very important to maintain the crystallization water at the heptahydrate level during drying. In addition, when a large amount of surface water is present, crystals are transformed into an amorphous form and agglomeration occurs, so loss due to agglomeration occurs during the purification process.
[4]
Due to the characteristics of these nucleic acids, drying has been carried out at a low temperature at room temperature as a method for removing the residual organic solvent in the crystallized nucleic acid, but there is a problem in that drying at a low temperature does not completely remove the residual organic solvent in the crystal. On the other hand, if drying is performed at a high temperature to completely remove the residual organic solvent in the crystal, it is advantageous to remove the organic solvent, but there is a problem in that the productivity of the nucleic acid crystal is deteriorated due to evaporation of the crystal water. .
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[5]
One object of the present application is a first step of crystallizing a nucleic acid into a solution containing a hydrophilic organic solvent; and a second step of drying the crystallized nucleic acid with air having a temperature of 30° C. or higher and 90° C. or lower, and a relative humidity of 40% or higher and 90% or lower.
means of solving the problem
[6]
In order to achieve the above object, one aspect of the present application is a first step of crystallizing a nucleic acid into a solution containing a hydrophilic organic solvent; and a second step of drying the crystallized nucleic acid with air having a temperature of 30° C. or higher and 90° C. or lower, and a relative humidity of 40% or higher and 90% or lower.
[7]
The nucleic acid purification method of the present application includes a first step of crystallizing the nucleic acid into a solution containing a hydrophilic organic solvent. In the present application, the crystallization method is not particularly limited as long as it uses a solution containing a hydrophilic organic solvent.
[8]
As used herein, the term “nucleic acid” refers to a compound consisting of a base, a sugar, and a phosphoric acid. Specifically, in the present application, the nucleic acid may be any one or more selected from the group consisting of 5'-guanylic acid (5'-GMP) and 5'-inosine acid (5'-IMP), and more specifically, the nucleic acid is 5' -Guanilic acid (5'-GMP) may be, but is not limited thereto.
[9]
In the present application, the nucleic acid is meant to include both the salt (salt) of the nucleic acid compound or the hydrate (hydrate) form of the salt.
[10]
As used herein, the term “salt” refers to a form in which a cation and an anion are combined by electrostatic attraction, and is typically a metal salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, and basicity. or a salt with an acidic amino acid. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine salts with, etc.; salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid, or the like.
[11]
As used herein, the term “hydrate” refers to a form in which water is bound to a compound, and water contained when the hydrate is a crystal is called crystal water.
[12]
That is, in the present application, the nucleic acid refers to a nucleic acid compound, a salt thereof, or a hydrate of the salt. Specifically, the nucleic acid is 5'-guanylic acid (5'-GMP) and 5'-inosine acid (5'-IMP). ), may be any one or more selected from the group consisting of 5'-guanylate disodium (5'-GMP 2Na) and 5'-inosodium disodium (5'-IMP 2Na), which are salts of, and more specifically, 5'-guanylate 2Na It may be sodium (5'-GMP 2Na), but is not limited thereto.
[13]
In addition, specifically, the nucleic acid may be a hydrate of 5'-guanylate disodium (5'-GMP 2Na) and 5'-inosine disodium (5'-IMP 2Na). Specifically, the nucleic acid may include 5'-guanylate disodium heptahydrate (5'-GMP 2Na 7H 2 O) or 5'-disodium inosinate (5'-IMP 2Na) 7.5 hydrate, but is not necessarily limited thereto it is not
[14]
As used herein, the term “hydropholic organic solvent” refers to an organic solvent exhibiting hydrophilic properties, and specifically, the organic solvent may be any one or more selected from the group consisting of methanol and ethanol, more specifically It may be methanol, but is not limited thereto.
[15]
The first step of the present application is specifically (i) adding a solution containing a hydrophilic organic solvent to the nucleic acid concentrate; (ii) cooling the nucleic acid concentrate to which the solution is added; (iii) separating the resulting nucleic acid crystal slurry by centrifugation; and (iv) washing the isolated nucleic acid crystal, but is not limited thereto.
[16]
In the present application, the hydrophilic organic solvent may be added at 1.0 RV or more and 1.5 RV or less compared to the nucleic acid concentrate, specifically, 1.0 RV or more and 1.5 RV or less, 1.1 RV or more 1.4 RV or less, 1.1 RV or more and 1.3 RV or less, 1.15 RV It may be added at or below 1.25 RV, but is not limited thereto.
[17]
In the present application, the cooling may be performed for 1 hour or more and 3 hours or less, and specifically, 1 hour or more and 3 hours or less, 1.5 hours or more and 2.5 hours or less, but is not limited thereto.
[18]
In the present application, the cooling may be performed at 20°C or more and 30°C or less, and specifically, at 20°C or more and 30°C or less, 22°C or more and 28°C or less, 23°C or more and 27°C or less, 24°C or more and 26°C or less. can be performed, but is not limited thereto.
[19]
In the present application, the centrifugation may be performed at 2000 rpm or more and 3000 rpm or less, but is not limited thereto.
[20]
The nucleic acid purification method of the present application includes a second step of drying the crystallized nucleic acid with air having a temperature of 30°C or higher and 90°C or lower, and a relative humidity of 40% or higher and 90% or lower.
[21]
The second step of the present application is specifically (a) adjusting the temperature and humidity of the air; And (b) drying the nucleic acid crystals obtained in the first step with temperature and humidity controlled air, but is not limited thereto.
[22]
In the present application, the air may have a temperature of 30°C or more and less than 60°C, and a relative humidity of 40% or more and 90% or less. Specifically, the temperature is 30 ℃ or more and less than 60 ℃, 30 ℃ or more 55 ℃ or less, 35 ℃ or more and 55 ℃ or less, 35 ℃ or more and 50 ℃ or less, 40 ℃ or more and 55 ℃ or less, 45 ℃ or more and 55 ℃ or less, 45 ℃ It may be greater than 55°C or less, 45°C or more and less than 60°C, or 50°C or more and less than 60°C, wherein the relative humidity is 40% or more and 90% or less, 55% or more and 85% or less, 50% or more and 80% or less, 40% or more. 60% or less, 45% or more and 55% or less, 80% or more and 90% or less, but is not limited thereto.
[23]
In the present application, the air may have a temperature of 60°C or more and 90% or less, and a relative humidity of 70% or more and 90% or less. Specifically, the temperature may be 60 ℃ or more and 90 ℃ or less, 60 ℃ or more and 85 ℃ or less, 60 ℃ or more and 80 ℃ or less, 65 ℃ or more and 90 ℃ or less, 65 ℃ or more and 80 ℃ or less, 65 ℃ or more and 75 ℃ or less, The relative humidity may be 70% or more and 90% or less, 75% or more and 90% or less, 80% or more and 90% or less, but is not limited thereto.
[24]
In the present application, the drying may be performed for 2 hours or more and 7 hours or less. Specifically, it may be 2 hours or more and 7 hours or less, 2.5 hours or more and 6.5 hours or less, 3 hours or more and 6 hours or less, but is not limited thereto.
[25]
In the present application, the drying method is not particularly limited as long as it uses high-humidity hot air, that is, air with temperature and relative humidity controlled. Specifically, the drying may be performed with a dryer capable of controlling the humidity of the air, but is not limited thereto.
[26]
The dryer used in this application is composed of a temperature and humidity control device and a dryer chamber (FIG. 2). After supplying high-humidity hot air with temperature and humidity controlled by the temperature and humidity control device to the dryer chamber, a wet well is introduced The organic solvent and surface water in the nucleic acid crystal can be removed. 2 is a schematic diagram showing an example of a dryer usable in the present application.
Effects of the Invention
[27]
The nucleic acid purification method of the present application can completely remove the organic solvent remaining after nucleic acid crystallization using a hydrophilic organic solvent by drying with high humidity and hot air. In addition, the crystal water of the nucleic acid is maintained even during drying, and the hydrate structure is maintained, so that aggregation of the crystals does not occur, thereby showing an excellent yield.
[28]
In addition, since the above effects can be exhibited only by the high-humidity hot air drying step, there is also a cost reduction effect, so that it can be widely used for more economical purification of nucleic acids.
Brief description of the drawing
[29]
1 is a graph showing the loss of crystal water of nucleic acid crystals according to temperature.
[30]
2 is a schematic diagram showing an example of a dryer usable in the present application.
Modes for carrying out the invention
[31]
Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.
[32]
[33]
Experimental Example 1.
[34]
Experimental Example 1-1. Crystallization of Nucleic Acids
[35]
A concentrate was prepared in which 5'-disodium guanylate heptahydrate was present at a level of 250 g/L. A hydrophilic organic solvent corresponding to 1.2RV (Relative Volume) relative to the volume of the concentrate was added at a flow rate of 0.2RV/hr at 38°C for 6 hours. Cooling was performed to 25° C. for 2 hours from the time the input was completed, and the crystal slurry was separated through a centrifuge. At this time, centrifugation was performed at a speed of 2000 rpm to 3000 rpm, and an aqueous solution containing 50% of a hydrophilic organic solvent was used as the washing solution, and the crystals were washed by spraying at 2000 rpm. After washing was completed, a wet tablet of 5'-guanylate disodium heptahydrate having a moisture content of 30% was obtained.
[36]
[37]
Experimental Example 1-2. Drying of crystallized nucleic acids
[38]
The high-humidity hot air used for crystal drying was controlled using a humidity control device installed at the bottom of the dryer (FIG. 2). After heating low-temperature air to high temperature, the humidity was adjusted, and after stabilization through a humidity control device, high-humidity hot air was supplied to the dryer chamber. Thereafter, the wet crystal obtained in Experimental Example 1-1 was introduced to remove the organic solvent and surface water in the nucleic acid crystal. The temperature and humidity inside the dryer were observed using a thermo-hygrometer installed inside the chamber.
[39]
[40]
Experimental Example 2. Crystal Analysis
[41]
Experimental Example 2-1. Analysis of changes in organic solvent content
[42]
Gear: Hewlett 5890 parkard series 2
[43]
Column: Porapak q (waters associates, 6FT 1/8 in 80/100 packed column supelco)
[44]
Carrier gas: hydrogen, nitrogen
[45]
Detector Type: FLD
[46]
Oven temperature: 140℃
[47]
Sample inlet temperature: 150℃
[48]
Detector temperature: 175℃
[49]
Sample injection volume: 1 μL
[50]
[51]
In order to analyze the organic solvent content of the crystals, 1.0000 g of 5'-guanylate disodium heptahydrate crystals were quantified, placed in a 0.01 L volumetric flask, and diluted with ultrapure water to prepare a 100 g/L sample. Then, a methanol (or ethanol) standard reagent (JTbaker >99.0%) was prepared at 50 mg/L, the standard reagent was used as an external standard, and the sample was analyzed by gas chromatography (GC).
[52]
[53]
Experimental Example 2-2. Analysis of crystal water change through measurement of residual hydrate
[54]
To analyze the residual hydrate content, 20 mg of 5'-guanylate disodium heptahydrate crystals were quantified and placed in a thermogravimetric analyzer pan. Thereafter, the temperature of the thermal analyzer was increased from the initial temperature of 25°C to 300°C at a rate of 2°C/min to observe the weight change. At this time, the 5'-disodium guanylate heptahydrate crystals exhibited a weight change of 23.6% at about 200°C, which can be seen as a weight change due to evaporation of the heptahydrate, and the residual hydrate content was analyzed through this.
[55]
[56]
Experimental Example 2-3. Yield change analysis
[57]
In order to analyze the yield after drying, the loss occurred during drying was measured by measuring the weight of the fine powder collected in the bag filter mounted on the top of the dryer after drying was completed.
[58]
[59]
Experimental Example 3. Observation of changes in crystal water according to temperature and humidity conditions
[60]
Changes in the crystal water in the 5'-disodium guanylate heptahydrate wet crystal were observed while changing the temperature and humidity conditions in the dryer chamber through the humidity control device installed at the bottom of the dryer.
[61]
As a result, as shown in Table 1, the residual hydrate was 12.7% to 25.2% when dried under the conditions of a temperature of 30°C or more and 90°C or less and a relative humidity of 40% or more and 90% or less.
[62]
In particular, when drying under the conditions of a temperature of 30°C or higher and less than 60°C, a relative humidity of 40% or higher, or a temperature of 60°C or higher and 90°C or lower, and a relative humidity of 80% or higher, the residual hydrate is 21.4% to 25.2%, the above range It was confirmed that there was a significant amount of residual hydrate.
[63]
[64]
[Table 1]
　Moisture (%) Temperature (℃)
36 50 70
Relative Humidity (RH) 10 8.8 7.6 5.4
50 25.2 21.9 12.7
60 24 21.4 13.4
70 22.1 20.6 13.5
75 23.7 22.7 13.9
80 24 24 21.4
[65]
From the above results, it was found that the crystal water was maintained even during long-term treatment in the dryer under the temperature and humidity conditions. In addition, at the level of 23.6% (±2%), which is the theoretical amount of crystals of 5'-disodium guanylate heptahydrate, the hydrate structure was not deformed during the drying process, so the aggregation of the crystals did not occur, resulting in yield loss due to aggregation It was confirmed that this did not appear. In addition, it was confirmed that the crystallinity was excellent because there was no overdrying process.
[66]
Through this, when the temperature and relative humidity are within the range, the water of crystallization is maintained step by step and the organic solvent can be completely removed, and the hydrate structure is not deformed during the drying process, so that the crystal aggregation phenomenon does not occur, and the yield by agglomeration It was confirmed that there was no loss.
[67]
[68]
Example 1. Purification of Nucleic Acid I
[69]
After the temperature and humidity in the dryer chamber were kept constant at 35°C and 50% through a humidity control device installed at the bottom of the dryer, wet crystals of 5'-disodium guanylate heptahydrate were continuously introduced. At this time, the moisture in the wet crystal was about 30% of the surface water and the crystal water.
[70]
As a result, as shown in Table 2, the residual hydrate was 22.3% and the methanol content was 0ppm when dried for 6 hours under the conditions of a temperature of 34°C and a relative humidity of 48%.
[71]
[72]
[Table 2]
　 driving conditions Decision Analysis Results
Time Temperature and humidity inside the dryer exit DAMPER Moisture MEOH
Min temp RH temp % % ppm
0 36 43 36 50 31 808
15 35 48 34 50 21 80.2
60 35 48 35 65 21.5 24.4
120 34 50 34 60 21.1 21.3
180 34 52 34 70 21.4 15.7
360 34 52 34 70 22.3 ND
[73]
From the above results, it was found that, under the temperature and humidity conditions, crystallized water was maintained step by step during drying and the complete removal of methanol was possible. In addition, since the hydrate structure was not deformed during the drying process, crystal aggregation did not occur, and it was confirmed that there was no yield loss due to aggregation.
[74]
[75]
Example 2. Purification of Nucleic Acid II
[76]
After the temperature and humidity in the dryer chamber were kept constant at 55°C and 60% through a humidity control device installed at the bottom of the dryer, a wet crystal of 5'-disodium guanylate heptahydrate was continuously introduced. At this time, the moisture in the wet crystal was about 30% of the surface water and the crystal water.
[77]
As a result, as shown in Table 3, the residual hydrate was 23% and the methanol content was 0ppm when dried for 3 hours at a temperature of 55°C and a relative humidity of 60%.
[78]
[79]
[Table 3]
　 driving conditions Decision Analysis Results
Time Temperature and humidity inside the dryer exit DAMPER Moisture MEOH
Min temp RH temp % % ppm
0 55 42 55 50 30 812
15 56 50 56 50 20 70
30 58 55 58 50 19 20
60 55 52 55 60 20 17
120 55 72 55 65 21 12
180 55 71 55 60 23 ND
[80]
From the above results, it was found that, under the temperature and humidity conditions, crystallized water was maintained step by step during drying and the complete removal of methanol was possible. In addition, since the hydrate structure was not deformed during the drying process, crystal aggregation did not occur, and it was confirmed that there was no yield loss due to aggregation.
[81]
[82]
Example 3. Purification of Nucleic Acid III
[83]
After the temperature and humidity in the dryer chamber were kept constant at 70°C and 80% through a humidity control device installed at the bottom of the dryer, wet crystals of 5'-disodium guanylate heptahydrate were continuously introduced. At this time, the moisture in the wet crystal was about 30% of the surface water and the crystal water.
[84]
As a result, as shown in Table 4, the residual hydrate was 23% and the methanol content was 0ppm when dried for 3 hours at a temperature of 70°C and a relative humidity of 80%.
[85]
[86]
[Table 4]
　 driving conditions Decision Analysis Results
Time Temperature and humidity inside the dryer exit DAMPER Moisture MEOH
Min temp RH temp % % ppm
0 68 77 68 50 60 809
30 70 79 70 50 23 20
60 71 80 71 50 22 18
90 71 80 71 50 22 15
120 71 80 71 50 23 12
150 71 80 71 50 21 6
180 70 81 70 60 23 ND
18hr 70 80 70 60 24 ND
[87]
From the above results, it was found that, under the temperature and humidity conditions, crystallized water was maintained step by step during drying and the complete removal of methanol was possible. In addition, since the hydrate structure was not deformed during the drying process, crystal aggregation did not occur, and it was confirmed that there was no yield loss due to aggregation.
[88]
[89]
Comparative Example 1. Purification of Nucleic Acid IV
[90]
After maintaining a constant temperature in the dryer chamber at 37 ° C through a humidity control device installed at the bottom of the dryer (relative humidity unadjusted dry air; humidity level of 13%), 5'-disodium guanylate heptahydrate wet crystals were continuously introduced. . At this time, the moisture in the wet crystal was about 30% of the surface water and the crystal water.
[91]
As a result, as shown in Table 5, the residual hydrate was 13% and the methanol content was 9ppm when dried for 3 hours under conditions of temperature 37°C and unadjusted relative humidity (13% level).
[92]
[93]
[Table 5]
　 driving conditions Decision Analysis Results
Time Temperature and humidity inside the dryer exit DAMPER Moisture MEOH
Min temp RH temp % % ppm
0 36 10 36 50 30 810
15 37 12 37 50 19 65
30 38 12 38 50 18 25
60 36 15 36 50 17 21
120 36 15 36 50 15 16
180 36 13 36 50 13 9
[94]
From the above results, it was found that the complete removal of methanol was impossible under the temperature and humidity conditions. In addition, it was confirmed that the amount of crystallization was 13%, which caused a loss of about 45% compared to the theoretical amount of crystallization, which resulted in poor crystallinity and impossible to achieve quality due to insufficient amount of hydrate.
[95]
[96]
Comparative Example 2. Purification of nucleic acids V
[97]
After maintaining a constant temperature and humidity at 70°C and 50% through a humidity control device installed at the bottom of the dryer, wet crystals of 5'-disodium guanylate heptahydrate were continuously introduced. At this time, the moisture in the wet crystal was about 30% of the surface water and the crystal water.
[98]
As a result, as shown in Table 6, the residual hydrate was 13% and the methanol content was 0ppm when dried for 3 hours at a temperature of 37°C and a relative humidity of 50%.
[99]
[100]
[Table 6]
　 driving conditions Decision Analysis Results
Time Temperature and humidity inside the dryer exit DAMPER Moisture MEOH
Min temp RH temp % % ppm
0 68 43 68 50 31 812
30 70 51 70 50 21 43
60 71 48 71 50 21 29
90 71 48 71 50 18 18
120 71 51 71 50 17 11
150 71 43 71 50 14 9
180 70 51 70 50 13 ND
[101]
Through the above results, it can be seen that the complete removal of methanol is possible under the temperature and humidity conditions. However, it was confirmed that the amount of crystallization was 13%, which caused a loss of about 45% compared to the theoretical amount of crystallization.
[102]
From the above results, in the temperature and humidity range of the present application, that is, the temperature 30 ℃ or more and 90 ℃ or less and the relative humidity 40% or more and 90% or less conditions, specifically, the humidity of 40% or more and 90% or less at the temperature of 30 ℃ or more and less than 60 ℃ As a result, it was found that at a temperature of 60°C or higher and 90°C or lower, when maintained at a humidity of 70% or higher and 90% or lower, crystal water was maintained, methanol was completely removed, and there was no yield loss due to crystal aggregation. . In addition, it was found that when out of the above range, the crystal water evaporated or the organic solvent could not be completely removed according to the decrease in humidity.
[103]
From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.
Claims
[Claim 1]
A first step of crystallizing the nucleic acid into a solution containing a hydrophilic organic solvent; and a second step of drying the crystallized nucleic acid with air having a temperature of 30°C or more and 90% or less, and a relative humidity of 40% or more and 90% or less.
[Claim 2]
The method of claim 1, wherein the nucleic acid comprises at least one selected from the group consisting of 5'-disodium guanylate and disodium 5'-inosinate.
[Claim 3]
The method of claim 1, wherein the nucleic acid comprises 5'-disodium guanylate heptahydrate or 5'-disodium inosinate 7.5 hydrate.
[Claim 4]
The method of claim 1, wherein the hydrophilic organic solvent is at least one selected from the group consisting of methanol and ethanol.
[Claim 5]
The method of claim 1, wherein the air has a temperature of 30°C or more and less than 60°C, and a relative humidity of 40% or more and 90% or less.
[Claim 6]
The method of claim 1, wherein the air has a temperature of 70°C or more and 90% or less, and a relative humidity of 70% or more and 90% or less.