Nucleic acid purification methods

Abstract

To provide a method for purifying nucleic acid.SOLUTION: Provided is a method for purifying nucleic acid comprising: a first step of crystallizing nucleic acid in a solution containing a hydrophilic organic solvent; and a second step of drying the crystallized nucleic acid in air at a temperature of 45°C or higher to 60°C or lower and a relative humidity of 40% or more to 90% or less, the nucleic acid containing 5'-disodium guanylate 7-hydrate or 5'-disodium inosinate 7.5 hydrate.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] This application relates to a method for purifying nucleic acids. Specifically, it relates to a method for purifying nucleic acids, which includes a first step of crystallizing the nucleic acids with a solution containing a hydrophilic organic solvent, and a second step of drying the crystallized nucleic acids with hot and humid air.

Background Art

[0002] As a method for crystallizing nucleic acids, a method using a hydrophilic organic solvent is mainly used. However, in nucleic acids used for food, due to regulations on the residual amount of organic solvents in various countries, the removal of organic solvents in the final product is a very important issue. Examples of hydrophilic organic solvents generally used in nucleic acid crystallization include ethanol, methanol, etc. In particular, methanol has strict residual regulations, so complete removal is required. Nevertheless, the method of using an organic solvent as a nucleic acid crystallization method (Patent Document 1) is still common at present. In relation to this, in addition to the above-mentioned patent document, Patent Documents 2, 3, 4, 5, 6, etc. also disclose methods for crystallizing nucleic acids using organic solvents.

[0003] Nucleic acid crystals, especially sodium 5'-guanylate heptahydrate crystals existing in the form of heptahydrate, lose crystal water even at a relatively low temperature around room temperature. When the temperature is raised from room temperature to 80°C over about 30 minutes, the amount decreases by about 70% from heptahydrate to 2.5 hydrate (Figure 1). Such loss of hydrate in sodium 5'-guanylate heptahydrate crystals reduces the crystallinity of the crystals, causing crystal deterioration and changes in crystal form. Therefore, it is very important to maintain the crystal water at the heptahydrate level during drying. Also, when a large amount of surface water is present, the crystals deform into an amorphous form and aggregation occurs, resulting in losses due to aggregation during the purification process.

[0004] Due to these characteristics of nucleic acids, drying at low temperatures, around room temperature, has been used to remove residual organic solvents from crystallized nucleic acids. However, low-temperature drying has the problem of not being able to completely remove residual organic solvents from the crystals. On the other hand, drying at high temperatures to completely remove residual organic solvents from the crystals is advantageous in removing organic solvents, but it has the problem of reducing the product quality of the nucleic acid crystals due to the evaporation of crystal water. Therefore, an alternative method is needed. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Korean Registered Patent Publication No. 10-0051324 [Patent Document 2] Korean Registered Patent Publication No. 10-0083595 [Patent Document 3] Chinese Patent No. 100395256 Specification [Patent Document 4] Specification of Chinese Patent No. 101863943 [Patent Document 5] Korean Registered Patent Publication No. 10-0025552 [Patent Document 6] Korean Registered Patent Publication No. 10-0117428 [Overview of the project] [Problems that the invention aims to solve]

[0006] This application aims to provide a method for purifying nucleic acids, comprising a first step of crystallizing nucleic acids in a hydrophilic organic solvent-containing solution, and a second step of drying the crystallized nucleic acids in air at a temperature of 30°C to 90°C and a relative humidity of 40% to 90%. [Means for solving the problem]

[0007] To achieve the above objective, one aspect of this application provides a method for crystallizing nucleic acids in a hydrophilic organic solvent-containing solution. The present invention provides a method for purifying nucleic acids, comprising a first step of crystallization and a second step of drying the crystallized nucleic acid in air at a temperature of 30°C to 90°C and a relative humidity of 40% to 90%.

[0008] The nucleic acid purification method of this application includes a first step of crystallizing nucleic acids in a hydrophilic organic solvent-containing solution. The crystallization method in this application may be any method that uses a hydrophilic organic solvent-containing solution.

[0009] In this application, "nucleic acid" means a compound consisting of a base, a sugar, and a phosphate. Specifically, the nucleic acid in this application is at least one selected from the group consisting of 5'-guanylic acid (5'-GMP) and 5'-inosinic acid (5'-IMP), and more specifically, the nucleic acid is 5'-guanylic acid (5'-GMP), but is not limited to these.

[0010] In this application, the nucleic acid is a salt of a nucleic acid compound, or a hydrate of the salt. It encompasses all forms.

[0011] In this application, "salt" refers to a form in which a cation and anion are bonded by electrostatic attraction, and typically includes metal salts, salts with organic bases, salts with inorganic acids, salts with basic or acidic amino acids, etc. For example, examples of metal salts include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), and aluminum salts. Examples of salts with organic bases include salts with triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylenediamine. Examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Examples of salts with organic acids include 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, and p-toluenesulfonic acid. Examples of salts with basic amino acids include salts with arginine, lysine, and ornithine. Examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid.

[0012] In this application, "hydrate" refers to a form in which water is bonded to the compound. If the hydrate is crystalline, the water it contains is called crystal water.

[0013] In other words, the term "nucleic acid" in this application refers collectively to nucleic acid compounds, their salts, or hydrates of said salts. Specifically, the nucleic acid is at least one selected from the group consisting of 5'-guanylate disodium (5'-GMP 2Na) and 5'-inosinate disodium (5'-IMP 2Na), which are salts of 5'-guanylate (5'-GMP) and 5'-inosinate (5'-IMP). More specifically, it is 5'-guanylate disodium (5'-GMP 2Na), but is not limited to these.

[0014] More specifically, the nucleic acid is a hydrate of 5'-disodium guanylate (5'-GMP 2Na) and 5'-inosinate (5'-IMP 2Na). More specifically, the nucleic acid is 5'-disodium guanylate heptahydrate (5'-GMP 2Na 7H2O) or 5'-inosinate The substance is disodium (5'-IMP 2Na) 7.5-hydrate, but is not necessarily limited to this.

[0015] In this application, "hydrophobic organic solvent" means hydrophilic This refers to organic solvents that indicate quality, and specifically, the organic solvents are methanol and ethanol. At least one selected from the following group, more specifically methanol, but not limited to these.

[0016] The first step of this application specifically includes, but is not limited to, (i) adding a hydrophilic organic solvent-containing solution to a nucleic acid concentrate, (ii) cooling the nucleic acid concentrate to which the solution has been added, (iii) separating the resulting nucleic acid crystal slurry by centrifugation, and (iv) washing the separated nucleic acid crystals.

[0017] In this application, the hydrophilic organic solvent is added to the nucleic acid concentrate in an amount of 1.0 RV or more and 1.5 RV or less, specifically in amounts of 1.0 RV or more and 1.5 RV or less, 1.1 RV or more and 1.4 RV or less, 1.1 RV or more and 1.3 RV or less, and 1.15 RV or more and 1.25 RV or less, but is not limited to these amounts.

[0018] In this application, the cooling is performed for a period of 1 hour or more and 3 hours or less, specifically 1 hour or more and 1.5 hours or more and 2.5 hours or less, but is not limited to these periods.

[0019] In this application, the cooling is carried out at 20°C or higher and 30°C or lower. Specifically, it is carried out at 20°C or higher and 30°C or lower, 22°C or higher and 28°C or lower, 23°C or higher and 27°C or lower, 24°C or higher and 26°C or lower, but is not limited thereto.

[0020] In this application, the centrifugation is carried out at 2000 rpm or higher and 3000 rpm or lower, but is not limited thereto.

[0021] The nucleic acid purification method of this application includes a second step of drying the crystallized nucleic acid with air at a temperature of 30°C or higher and 90°C or lower and a relative humidity of 40% or higher and 90% or lower.

[0022] Specifically, the second step of this application includes: (a) a step of adjusting the temperature and humidity of the air; and (b) a step of drying the nucleic acid crystal obtained in the first step with the air whose temperature and humidity have been adjusted, but is not limited thereto.

[0023] In this application, the air may have a temperature of 30°C or higher and less than 60°C and a relative humidity of 40% or higher and 90% or lower.Specifically, the temperature is 30°C or higher and less than 60°C, 30°C or higher and 55°C or lower, 35°C or higher and 55°C or lower, 35°C or higher and 50°C or lower, 40°C or higher and 55°C or lower, 45°C or higher and 55°C or lower, more than 45°C and 55°C or lower, 45°C or higher and less than 60°C, or 50°C or higher and less than 60°C, and the relative humidity is 40% or higher and 90% or lower, 55% or higher and 85% or lower, 50% or higher and 80% or lower, 40% or higher and 60% or lower, 45% or higher and 55% or lower, 80% or higher and 90% or lower, but is not limited thereto.

[0024] In this application, the air may have a temperature of 60°C or higher and 90°C or lower and a relative humidity of 70% or higher and 90% or lower.Specifically, the temperature is 60°C or higher and 90°C or lower, 60°C or higher and 85°C or lower, 60°C or higher and 80°C or lower, 65°C or higher and 90°C or lower, 65°C or higher and 80°C or lower, 65°C or higher and 75°C or lower, and the relative humidity is 70% or higher and 90% or lower, 75% or higher and 90% or lower, 80% or higher and 90% or lower, but is not limited thereto.

[0025] In this application, the drying is carried out over a period of 2 hours to 7 hours. Specifically, this includes, but is not limited to, 2 hours to 7 hours, 2.5 hours to 6.5 hours, and 3 hours to 6 hours.

[0026] In this application, the drying method may be any method that uses high-humidity hot air, that is, air whose temperature and relative humidity have been controlled. Specifically, the drying method involves controlling the humidity of the air. This is done using a dryer capable of doing so, but is not limited to this.

[0027] The dryer used in this application consists of a temperature and humidity control device and a dryer chamber (Figure 2). High-humidity hot air, whose temperature and humidity are controlled by the temperature and humidity control device, is supplied to the dryer chamber, and then a wet tablet is injected, which removes organic solvents and surface water from within the nucleic acid crystal. Figure 2 is a schematic diagram showing an example of the dryer used in this application. [Effects of the Invention]

[0028] The nucleic acid purification method described in this application can completely remove residual organic solvents after nucleic acid crystallization using hydrophilic organic solvents by high-humidity hot air drying. Furthermore, since the water of crystallization of nucleic acids is maintained during drying, and the structure of the hydrate is preserved, crystal aggregation does not occur, resulting in excellent yield.

[0029] Furthermore, since the above effects can be obtained solely through the high-humidity hot air drying step, there is also a cost reduction effect, making nucleic acid purification more economical. [Brief explanation of the drawing]

[0030] [Figure 1] This graph shows the loss of crystal water in nucleic acid crystals due to temperature. [Figure 2] This is a schematic diagram showing an example of a dryer used in this application. [Modes for carrying out the invention]

[0031] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention, and the present invention is not limited to them.

[0032] Experimental Example 1. Experimental Example 1-1. Crystallization of nucleic acids A concentrated solution containing 250 g / L of disodium 5'-guanylate heptahydrate was prepared. Hydrophilic water equivalent to 1.2 RV (Relative Volume) relative to the volume of the concentrated solution was added. A hydrophilic organic solvent was added at a flow rate of 0.2 RV / hr over 6 hours at 38°C. After the addition was complete, the mixture was cooled to 25°C over 2 hours, and the crystal slurry was separated by centrifugation. Here, centrifugation was performed at a speed of 2000 rpm to 3000 rpm, and the crystals were washed by spray treatment using an aqueous solution containing 50% hydrophilic organic solvent at 2000 rpm. After washing, wet tablets of 5'-disodium guanylate heptahydrate with a water content of 30% were obtained.

[0033] Experimental Example 1-2. Drying of Crystallized Nucleic Acids The high-humidity hot air used for crystal drying was controlled using a humidity control device installed in the lower section of the dryer (Figure 2). Low-temperature air was heated to a high temperature, and then its humidity was adjusted before use. After stabilization using the humidity control device, the high-humidity hot air was supplied to the dryer chamber. Subsequently, the wet tablets obtained in Experimental Example 1-1 were injected to remove organic solvents and surface water from within the nucleic acid crystals. The temperature and humidity inside the dryer were monitored using a constant temperature and humidity meter installed inside the chamber.

[0034] Experimental Example 2. Crystal Analysis Experimental Example 2-1. Analysis of changes in organic solvent content Equipment: Hewlett 5890 parkard series 2 Column: Porapak q (waters associates, 6FT 1 / 8 in 80 / 100 packed column supelco) Carrier gas: Hydrogen, Nitrogen Detector type: FLD Oven temperature: 140℃ Temperature of the sample injection port: 150℃ Detector temperature: 175℃ Sample injection volume: 1 μL

[0035] To analyze the organic solvent content of the crystals, 1.0000 g of 5'-disodium guanylate heptahydrate crystals were weighed and placed in a 0.01 L flask, and diluted with ultrapure water to prepare a 100 g / L sample. Subsequently, a 50 mg / L methanol (or ethanol) standard reagent (JTbaker >99.0%) was prepared, and the sample was analyzed by gas chromatography (GC) using this standard reagent as an external standard.

[0036] Experimental Example 2-2. Analysis of changes in water of crystallization by measurement of residual hydrates. To analyze the residual hydrate content, 20 mg of 5'-disodium guanylate heptahydrate crystals were weighed and placed in a thermogravimetric analyzer pan. The thermogravimetric analyzer temperature was then increased from the initial temperature of 25°C to 300°C at a rate of 2°C / min, and the weight change was observed. At approximately 200°C, the 5'-disodium guanylate heptahydrate crystals showed a weight change of 23.6%. This can be attributed to the evaporation of the heptahydrate. The residual hydrate content was analyzed in this manner.

[0037] Experimental Example 2-3. Analysis of Yield Changes To analyze the yield after drying, the losses incurred during drying were measured by weighing the fine powder collected in a back filter mounted on the upper part of the dryer after the drying was complete.

[0038] Experimental Example 3: Observation of changes in water crystallization under temperature and humidity conditions. The temperature and humidity conditions inside the dryer chamber were changed using a humidity control device installed on the lower level of the dryer, and the changes in the water of crystallization within the 5'-disodium guanylate heptahydrate wet tablets were observed.

[0039] As a result, as shown in Table 1, when dried under conditions of a temperature between 30°C and 90°C and a relative humidity between 40% and 90%, the residual hydrate content was 12.7% to 25.2%.

[0040] In particular, when dried under conditions of a temperature between 30°C and 60°C and a relative humidity of 40% or higher, or a temperature between 60°C and 90°C and a relative humidity of 80% or higher, the residual hydrate content was 21.4% to 25.2%, confirming that the residual hydrate content was significantly higher within these ranges.

[0041] [Table 1]

[0042] These results indicate that under the above temperature and humidity conditions, crystal water can be retained even after prolonged processing inside the dryer. It was found that the crystals retained their properties. Furthermore, the crystal water content was at the theoretical level of 23.6% (±2%) for 5'-disodium guanylate heptahydrate crystals, and since the hydrate structure did not deform during the drying process, crystal aggregation did not occur, and it was confirmed that there was no yield loss due to aggregation. In addition, it was confirmed that the crystallinity was excellent because there was no over-drying process.

[0043] Therefore, it was confirmed that within the above temperature and relative humidity range, crystal water is maintained in stages, organic solvents can be completely removed, and the hydrate structure does not deform during the drying process, thus preventing crystal aggregation and eliminating yield loss due to aggregation. [Examples]

[0044] Nucleic acid purification I A humidity control device installed in the lower section of the dryer kept the temperature and humidity inside the dryer chamber constant at 35°C and 50%, and 5'-guanylate disodium heptahydrate wet tablets were continuously injected. At this point, the moisture content inside the wet tablets, including surface water and crystal water, was approximately 30%.

[0045] As a result, as shown in Table 2, when dried for 6 hours under conditions of 34°C and 48% relative humidity, the residual hydrate content was 22.3% and the methanol content was 0 ppm.

[0046] [Table 2]

[0047] These results indicate that under the above temperature and humidity conditions, crystal water is maintained in stages during drying, allowing for complete removal of methanol. Furthermore, since the hydrate structure does not deform during the drying process, crystal aggregation does not occur, and no yield loss due to aggregation is observed. [Examples]

[0048] Nucleic acid purification II A humidity control device installed in the lower section of the dryer kept the temperature and humidity inside the dryer chamber constant at 55°C and 60%, and 5'-disodium guanylate heptahydrate wet tablets were continuously injected. At this point, the moisture content inside the wet tablets, including surface water and crystal water, was approximately 30%.

[0049] As a result, as shown in Table 3, when dried for 3 hours under conditions of 55°C and 60% relative humidity, the residual hydrate was 23% and the methanol content was 0 ppm.

[0050] [Table 3]

[0051] These results indicate that under the above temperature and humidity conditions, crystal water is maintained in stages during drying, allowing for complete removal of methanol. Furthermore, since the hydrate structure does not deform during the drying process, crystal aggregation does not occur, and no yield loss due to aggregation is observed. [Examples]

[0052] Nucleic acid purification III A humidity control device installed on the lower level of the dryer kept the temperature and humidity inside the dryer chamber constant at 70°C and 80%, and 5'-disodium guanylate heptahydrate wet tablets were continuously injected. At this point, the moisture content inside the wet tablets, including surface water and crystal water, was approximately 30%.

[0053] As a result, as shown in Table 4, when dried for 3 hours under conditions of 70°C and 80% relative humidity, the residual hydrate was 23% and the methanol content was 0 ppm.

[0054] [Table 4]

[0055] These results indicate that under the above temperature and humidity conditions, crystal water is maintained in stages during drying, allowing for complete removal of methanol. Furthermore, since the hydrate structure does not deform during the drying process, crystal aggregation does not occur, and no yield loss due to aggregation is observed.

[0056] Comparative Example 1. Nucleic Acid Purification IV A humidity control device installed on the lower level of the dryer kept the temperature inside the dryer chamber constant at 37°C (relative humidity unadjusted, dry air: humidity level 13%), and 5'-disodium guanylate heptahydrate wet tablets were continuously injected. At this point, the moisture content inside the wet tablets, including surface water and crystal water, was approximately 30%.

[0057] As a result, as shown in Table 5, when dried for 3 hours at a temperature of 37°C and with no relative humidity adjustment (13% level), the residual hydrate was 13% and the methanol content was 9 ppm.

[0058] [Table 5]

[0059] These results indicate that methanol cannot be completely removed under the above temperature and humidity conditions. Furthermore, it was found that the crystal water content was at a level of 13%, resulting in a loss of approximately 45% compared to the theoretical amount of crystal water. This led to a decrease in crystallinity, and it was confirmed that the quality could not be achieved due to insufficient hydrate content.

[0060] Comparative Example 2. Purification of nucleic acids V A humidity control device installed on the lower level of the dryer kept the temperature and humidity inside the dryer chamber constant at 70°C and 50%, and wet tablets of 5'-disodium guanylate heptahydrate were continuously injected. At this point, the moisture content inside the wet tablets, including surface water and crystal water, was approximately 30%.

[0061] As a result, as shown in Table 6, when dried for 3 hours under conditions of 37°C and 50% relative humidity, the residual hydrate was 13% and the methanol content was 0 ppm.

[0062] [Table 6]

[0063] These results indicate that methanol can be completely removed under the above temperature and humidity conditions. However, the crystal water content is at a level of 13%, resulting in a loss of approximately 45% compared to the theoretical amount of crystal water. This leads to reduced crystallinity and insufficient hydrate, which prevents the desired quality from being achieved.

[0064] These results show that, within the temperature and humidity range specified in this application, namely a temperature of 30°C to 90°C and a relative humidity of 40% to 90%, specifically, maintaining a humidity of 40% to 90% at temperatures between 30°C and 60°C, and a humidity of 70% to 90% at temperatures between 60°C and 90°C, crystal water is maintained, methanol can be completely removed, and yield loss due to crystal aggregation does not occur. Furthermore, it was found that if the above range is deviated, the crystal water evaporates due to the decrease in humidity, and the organic solvent is not completely removed.

[0065] From the above description, those skilled in the art in which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. The first step involves crystallizing nucleic acids in a hydrophilic organic solvent-containing solution, The process includes a second step of drying the crystallized nucleic acid in air at a temperature greater than 50°C and 60°C or less, and a relative humidity of 40% or more and 90% or less. A method for purifying nucleic acids, wherein the nucleic acid comprises 5'-disodium guanylate heptahydrate or 5'-inosinate disodium heptahydrate.

2. The method for purifying nucleic acids according to claim 1, wherein the hydrophilic organic solvent is at least one selected from the group consisting of methanol and ethanol.

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