RU2797970C1 - Nucleic acid purification method - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a composition containing a highly concentrated inhibitor of alpha-1 proteinase (A1PI), and a method of its preparation. The method involves the use of single-pass filtration in a tangential flow.

EFFECT: invention is effective for the treatment of conditions associated with the treatment of diseases associated with deficiency of A1PI, or diseases in which it is beneficial to increase the level of A1PI.

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Description

Technical field

The invention relates to a method for purifying a nucleic acid, in particular a method for purifying a nucleic acid, which includes a first step of crystallizing the nucleic acid using a solution containing a hydrophilic organic solvent and a second step of drying the crystallized nucleic acid with hot air with high humidity.

State of the art

As methods for crystallizing nucleic acids, a method using a hydrophilic organic solvent is generally used. In the case of nucleic acids used in foodstuffs, the removal of organic solvents in the final products is an essential issue in view of the rules regarding the residual amount of organic solvents in each country. Hydrophilic organic solvents commonly used for nucleic acid crystallization include ethanol, methanol and the like. In particular, complete removal of methanol is required because the control of residual methanol is stringent. However, the organic solvent method (Korean Patent No. 10-0051324) is still widely used as a nucleic acid crystallization method. In this regard, in addition to this patent, Korean patent No. 10-0083595, Chinese Patent No. 100395256 Chinese Patent No. 101863943 Korean Patent No. 10-0025552 and Korean Patent No. 10-0117428 etc. also describe methods for crystallizing nucleic acids using an organic solvent.

Nucleic acid crystals, especially crystals of guanosine 5'-monophosphate disodium salt heptahydrate, which exist as a heptahydrate, lose water of crystallization at low temperatures, such as room temperature, and there is a loss of about 70% when the heptahydrate is converted to 2,5-hydrate when the temperature rises from room temperature to 80°C for about 30 minutes (figure 1). This loss of water of hydration in the crystals of disodium guanosine-5'-monophosphate heptahydrate reduces the crystallinity of the crystals and causes the crystals to weaken and change the shape of the crystals, and therefore it is essential to maintain the water of crystallization at the heptahydrate level during drying. When there is a large amount of surface water, the crystals are transformed into an amorphous form, agglomeration occurs, and thus, losses due to the agglomeration phenomenon also occur in the purification process.

In view of these properties of nucleic acids, as a method for removing residual organic solvent in a crystallized nucleic acid, drying at low temperatures such as room temperature is carried out. However, there is a problem that in the case of drying the nucleic acid crystals at a low temperature, the residual organic solvent in the crystals is not completely removed. On the other hand, it is advantageous to carry out drying at a high temperature to completely remove the residual organic solvent in the crystals under organic solvent removal conditions, but there is a problem that the quality of the nucleic acid crystal product is reduced due to the evaporation of water of crystallization, and an alternative to this is needed.

Brief description of the invention

The object of the invention is to provide a method for purifying a nucleic acid, which includes a first step of crystallizing the nucleic acid using 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°C or less and relative humidity. 40% or more and 90% or less.

Each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this specification are within the scope of the invention. In addition, the scope of protection of the invention is not limited to the following specific description.

In addition, those skilled in the art will understand, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. In addition, these equivalents should be interpreted as being within the scope of the protection of the invention.

In addition, throughout this description, when a part is referred to as "comprising" an element, it should be understood that other elements may be additionally included, but other elements are not excluded, unless the opposite is specifically described.

The invention is described in detail below.

To solve this problem, in one aspect of the invention, a method is provided for purifying a nucleic acid, which includes a first step of crystallizing the nucleic acid using 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° C or less and relative humidity 40% or more and 90% or less.

The method for purifying a nucleic acid according to the invention includes the first step of crystallizing the nucleic acid using a solution containing a hydrophilic organic solvent. In the present invention, the crystallization method is not particularly limited as long as it is a method using a solution containing a hydrophilic organic solvent.

As used herein, the term "nucleic acid" refers to a compound consisting of a base, sugar and phosphoric acid. In particular, in this invention, the nucleic acid may be any one or more acids selected from the group consisting of guanosine 5'-monophosphate (5'-GMP) and inosine 5'-monophosphate (5'-IMP), more specifically, but not limited to guanosine-5'-monophosphate (5'-GMP).

In the present invention, nucleic acid is intended to include both salts of nucleic acid compounds and hydrated forms of salts.

The term "salt" as used herein refers to the form in which the cation and anion are bonded to each other by electrostatic attraction and may generally be a metal salt, organic base salt, inorganic acid salt, organic acid salt, basic or acidic salt. amino acid and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt or the like), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt or the like), aluminum salt or the like; the organic base salt may be a salt with triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, or the like; the inorganic acid salt may be a salt with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid or the like; the organic acid salt may be a salt 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, or etc.; the basic amino acid salt may be an arginine, lysine, ornithine salt or the like; and the acidic amino acid salt may be an aspartic acid salt, glutamic acid salt, or the like.

The term "hydrate" as used herein refers to the form in which water is associated with a compound, and the water contained therein is referred to as water of crystallization when the hydrate is a crystal.

In other words, in the present invention, a nucleic acid refers to a compound of a nucleic acid, a salt thereof, or a hydrate of a salt. In particular, the nucleic acid may be any one or more acids selected from the group consisting of guanosine 5'-monophosphate disodium salt (5'-GMP 2Na) and inosine 5'-monophosphate disodium salt (5'-IMP 2Na ), which are the salt of guanosine-5'-monophosphate (5'-GMP) and the salt of inosine-5'-monophosphate (5'-IMP), respectively. More specifically, the nucleic acid may be guanosine 5'-monophosphate (5'-GMP 2Na) disodium salt, but is not limited thereto.

In particular, the nucleic acid may be hydrates of guanosine 5'-monophosphate disodium salt (5-HMP 2Na) and inosine 5'-monophosphate disodium salt (5'-IMP 2Na). In particular, the nucleic acid may be guanosine 5'-monophosphate disodium heptahydrate (5'-GMP 2Na 7H ₂ O) or inosine 5'-monophosphate disodium 7,5-hydrate (5'-IMP 2Na 7.5H ₂ O), but is not limited to them.

As used herein, the term "hydrophilic organic solvent" refers to an organic solvent exhibiting hydrophilic properties. In particular, the organic solvent may be any one or more solvents selected from the group consisting of methanol and ethanol, more specifically methanol, but not limited to this.

The first step of the invention may specifically include the step of (i) adding a solution containing a hydrophilic organic solvent to the nucleic acid concentrate; step (ii) cooling the nucleic acid concentrate to which the solution is added; step (iii) separating the resulting suspension of nucleic acid crystals by centrifugation; and step (iv) washing the isolated nucleic acid crystals, but not limited to this.

In the present invention, the hydrophilic organic solvent can be added at 1.0 RV or more and 1.5 RV or less, in particular at 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, or 1.15 RV or more and 1.25 RV or less with respect to, but not limited to, the nucleic acid concentrate.

In the present invention, cooling can be performed for 1 hour or more and 3 hours or less, in particular, 1 hour or more and 3 hours or less, or 1.5 hours or more and 2.5 hours or less, but not limited to this.

In the present invention, cooling can be performed at 20°C or more and 30°C or less, in particular 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, or 24°C or more and 26°C or less, but not limited to this.

In the present invention, centrifugation can be performed at 2000 rpm or more and 3000 rpm or less, but is not limited to this.

The method for purifying nucleic acids according to the invention includes a second step of drying the crystallized nucleic acid with air having a temperature of 30°C or more and 90°C or less and a relative humidity of 40% or more and 90% or less.

The second step of the present invention may specifically include, but is not limited to, a step (a) of controlling the temperature and humidity of the air, and a step (b) of drying the nucleic acid crystals obtained in the first step with temperature and humidity controlled air.

In the present invention, 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. In particular, the temperature may be 30°C or more and less than 60°C, 30°C or more and 55°C or less, 35°C or more and 55°C or less, 35°C or more and 50°C. or less, 40°C or more and 55°C or less, 45°C or more and 55°C or less, 45°C or more and 55°C or less, 45°C or more and less than 60°C, or 50°C or more and less than 60°C, and the relative humidity can be 40% or more and 90% or less, 55% or more and 85% or less, 50% or more and 80% or less, 40% or more and 60% or less, 45% or more and 55% or less, or 80% or more and 90% or less, but temperature and relative humidity are not limited to this.

In the present invention, the air may have a temperature of 60°C or more and 90°C or less, and a relative humidity of 70% or more and 90% or less. In particular, the temperature may be 60°C or more and 90°C or less, 60°C or more and 85°C or less, 60°C or more and 80°C or less, 65°C or more and 90°C. C or less, 65°C or more and 80°C or less, or 65°C or more and 75°C or less, and the relative humidity can be 70% or more and 90% or less, 75% or more and 90% or less, or 80% or more and 90% or less, but temperature and relative humidity are not limited to this.

In the present invention, drying can be performed for 2 hours or more and 7 hours or less. In particular, the drying time 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 to this.

In the present invention, the drying method is not particularly limited if it is a method using hot air with high humidity, namely temperature and humidity controlled air. In particular, drying can be performed using a dryer capable of controlling air humidity, but is not limited to it.

The dryer used in the present invention consists of a temperature and humidity control device and a drying chamber (FIG. 2). Hot air with high humidity, the temperature and humidity of which is controlled by the temperature and humidity control device, can be supplied to the dryer chamber, and then wet crystals can be introduced to remove the organic solvent and surface water in the nucleic acid crystals. Fig. 2 is a schematic diagram illustrating an example of a dryer used in the present invention.

In the method for purifying nucleic acids according to the invention, the organic solvent remaining after the crystallization of the nucleic acid using the hydrophilic organic solvent can be completely removed by drying with hot air with high humidity. The water of crystallization in nucleic acids is retained during drying, the hydrate structure is retained, and crystal agglomeration does not occur, and thus an excellent yield is obtained.

Effects can only be exhibited by high humidity hot air drying, thus there is also a cost saving effect, and the nucleic acid purification method can be widely used to purify nucleic acids more economically.

Brief description of the drawings

Fig. 1 is a graph illustrating the loss of water of crystallization in nucleic acid crystals as a function of temperature; And

Fig. 2 is a schematic diagram illustrating an example of a dryer suitable for use in the present invention.

Detailed description of the invention

Hereinafter, the configuration and effects of the present invention are described in more detail with reference to illustrative embodiments. However, these illustrative embodiments are for illustrative purposes only, and the scope of protection of the invention is not intended to be limited to these illustrative embodiments.

Experimental example 1

Experimental Example 1-1. Nucleic acid crystallization

A concentrate was prepared in which guanosine 5'-monophosphate disodium salt heptahydrate was present at a concentration of about 250 g/l. A hydrophilic organic solvent corresponding to 1.2 RV (relative volume) relative to the volume of the concentrate was added at a flow rate of 0.2 RV/h at 38° C. for 6 hours. Cooling was performed to 25° C. within 2 hours from the time at which the feed was completed, and the crystal suspension was separated by centrifuge. At this time, centrifugation was carried out at a speed of 2000 rpm to 3000 rpm, an aqueous solution containing a hydrophilic organic solvent at a concentration of 50% was used as a washing solution, and the crystals were washed by spraying at 2000 rpm. After completion of washing, wet crystals of guanosine 5'-monophosphate disodium salt heptahydrate having a moisture content of 30% were obtained.

Experimental example 1-2. Drying of crystallized nucleic acid

The hot, high humidity air used to dry the crystals was controlled by a humidity control device installed at the bottom of the dryer (FIG. 2). After the low temperature air was heated to a high temperature, the humidity was adjusted to use it. After being stabilized by the humidity control device, hot air with high humidity was supplied to the dryer chamber. Thereafter, wet crystals obtained in Experimental Example 1-1 were supplied to remove the organic solvent and surface water contained in the nucleic acid crystals. The temperature and humidity inside the dryer was monitored using a thermohygrometer installed inside the chamber.

Experimental Example 2 Crystal Analysis

Experimental Example 2-1. Analysis of change in organic solvent content

Hardware: Hewlett Packard 5890 series 2

Column: Porapak q (Waters associates, 6FT 1/8 in (inch) 80/100 filled Supelco column)

Carrier gas: hydrogen, nitrogen

Detector type: FLD

Temperature of thermostatic cabinet: 140°C

Sample inlet temperature: 150°C

Detector temperature: 175°C

Sample injection volume: 1 μl

To analyze the organic solvent content of the crystals, disodium guanosine 5'-monophosphate heptahydrate crystals were accurately weighed at 1.0000 g, placed in a 0.01 L volumetric flask, and diluted with ultrapure water to obtain a 100 g/L sample. After that, a methanol (or ethanol) standard reagent (J.T. Baker > 99.0%) was prepared at a concentration of 50 mg/l, this standard reagent was used as an external standard, and the sample was analyzed by gas chromatography (GC).

Experimental Example 2-2. Analysis of the change in the amount of water of crystallization by measuring the residual hydrate

To analyze the residual hydrate content, crystals of disodium guanosine 5'-monophosphate heptahydrate were accurately weighed with a mass of 20 mg and placed in a thermogravimetric analyzer. Thereafter, the temperature of the thermogravimetric analyzer was raised from an initial temperature of 25° C. to 300° C. at a rate of 2° C./min to observe the change in mass. Meanwhile, in the crystals of disodium guanosine-5'-monophosphate heptahydrate, a mass change of 23.6% occurs in the region of about 200° C., this can be regarded as a mass change due to evaporation of the heptahydrate, and thus the residual hydrate content was analyzed.

Experimental example 2-3. Output Change Analysis

In order to analyze the yield after drying, the loss occurring during drying was measured by measuring the mass of the fine powder collected in the baghouse installed on top of the dryer after drying was completed.

Experimental Example 3 Observation of the change in the amount of water of crystallization depending on the state of temperature and humidity

Changes in the amount of water of crystallization in the wet crystals of disodium guanosine 5'-monophosphate heptahydrate under changing conditions of temperature and humidity in the dryer chamber were observed using a humidity control device installed at the bottom of the dryer.

As a result, as shown in Table 1, the residual hydrate content was found to be 12.7% to 25.2% when drying was carried out under conditions of temperature of 30° C. or more and 90° C. or less and a relative humidity of 40% or more. and 90% or less.

It was confirmed that the residual hydrate is 21.4% to 25.2%, especially when drying was carried out under the condition of a temperature of 30°C or more and less than 60°C and a relative humidity of 40% or more or a temperature of 60°C or more, and 90° C. or less and a relative humidity of 80% or more, and thus it was confirmed that significantly more residual hydrate was present in the above ranges.

With the help of the obtained results, it was found that the water of crystallization is retained during long-term processing in the dryer, as well as under the conditions of the specified temperature and humidity. Since the hydrate structure did not change shape during the drying process, since the water of crystallization was about 23.6% (±2%), which is the theoretical amount of water of crystallization in the crystals of guanosine 5'-monophosphate disodium salt heptahydrate, it was confirmed that the phenomenon of crystal agglomeration does not occur, and there is no yield loss due to agglomeration. In addition, it was confirmed that the crystallinity is also excellent since overdrying does not occur.

Due to this, it was confirmed that the water of crystallization is stored stepwise, and complete removal of the organic solvent is possible when the temperature and relative humidity are within the specified ranges, the phenomenon of crystal agglomeration does not occur, and there is no yield loss due to agglomeration, since the hydrate structure does not change shape in drying process.

Example 1 Purification of I Nucleic Acid

After the temperature and humidity in the dryer chamber were kept constant at 35° C. and 50%, wet crystals of disodium guanosine 5'-monophosphate heptahydrate were continuously introduced using a humidity control device installed at the bottom of the dryer. At this time, the humidity, including surface water and water of crystallization, in wet crystals was about 30%.

As a result, as shown in Table 2, the residual hydrate was 22.3% and the methanol content was 0 parts per million (ppm) when drying was carried out for 6 hours under the condition of a temperature of 34°C and a relative humidity of 48%.

From the results obtained, it was found that the water of crystallization is stored stepwise during drying, and complete removal of methanol is possible under the specified conditions of temperature and humidity. In addition, it was confirmed that the crystal agglomeration phenomenon does not occur and there is no yield loss due to agglomeration because the hydrate structure does not change shape during the drying process.

Example 2 Purification of II Nucleic Acid

After the temperature and humidity in the dryer chamber were kept constant at 55° C. and 60% using a humidity control device installed at the bottom of the dryer, wet crystals of disodium guanosine 5'-monophosphate heptahydrate were continuously introduced. At the same time, the moisture content, including surface water and water of crystallization, in wet crystals was about 30%.

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

From the results obtained, it was found that the water of crystallization is stored stepwise during drying, and complete removal of methanol is possible under the specified conditions of temperature and humidity. In addition, it was confirmed that the crystal agglomeration phenomenon does not occur and there is no yield loss due to agglomeration because the hydrate structure does not change shape during the drying process.

Example 3 Purification of III Nucleic Acid

After the temperature and humidity in the dryer chamber were kept constant at 70° C. and 80%, wet crystals of disodium guanosine 5'-monophosphate heptahydrate were continuously introduced using a humidity control device installed at the bottom of the dryer. At the same time, the moisture content, including surface water and water of crystallization, in wet crystals was about 30%.

As a result, as shown in Table 4, the residual hydrate was 23% and the methanol content was 0 ppm when drying was carried out for 3 hours under the condition of a temperature of 70° C. and a relative humidity of 80%.

From the results obtained, it was found that the water of crystallization is stored stepwise during drying, and complete removal of methanol is possible under the specified conditions of temperature and humidity. In addition, it was confirmed that the crystal agglomeration phenomenon does not occur and there is no yield loss due to agglomeration because the hydrate structure does not change shape during the drying process.

Comparative Example 1 Purification of IV Nucleic Acid

After the temperature (relative humidity - uncontrolled dry air; humidity about 13%) in the drying chamber was set constant at 37°C using a humidity control device installed at the bottom of the dryer, wet crystals of guanosine-5 disodium salt heptahydrate were continuously introduced '-monophosphate. At this time, the humidity, including surface water and water of crystallization, in wet crystals was about 30%.

As a result, as shown in Table 5, the residual hydrate was 13% and the methanol content was 9 ppm when drying was carried out for 3 hours at a temperature of 37° C. and an uncontrolled relative humidity (about 13%).

From these results, it was determined that complete removal of methanol was not possible under the indicated conditions of temperature and humidity. In addition, it was confirmed that the crystallinity is lower because the water of crystallization is about 13%, and there is a loss of about 45% based on the theoretical amount of water of crystallization, and the quality cannot be achieved due to insufficient hydrate.

Comparative Example 2 Purification of V Nucleic Acid

After the temperature and humidity in the dryer chamber were kept constant at 70° C. and 50%, wet crystals of disodium guanosine 5'-monophosphate heptahydrate were continuously introduced using a humidity control device installed at the bottom of the dryer. At the same time, the moisture content, including surface water and water of crystallization, in wet crystals was about 30%.

As a result, as shown in Table 6, the residual hydrate was 13% and the methanol content was 0 ppm when drying was carried out for 3 hours under the condition of a temperature of 70° C. and a relative humidity of 50%.

From the results, it was found that complete removal of methanol is possible under the specified conditions of temperature and humidity. However, it was confirmed that the crystallinity is lower because the water of crystallization is about 13%, and there is a loss of about 45% based on the theoretical amount of water of crystallization, and the quality cannot be achieved due to insufficient hydrate.

From the results described above, it was found that the water of crystallization is retained, the methanol is completely removed, and there is no loss of yield due to crystal agglomeration while maintaining the temperature and humidity ranges according to the invention, namely the temperature conditions of 30°C or more and 90°C or less and relative humidity of 40% or more and 90% or less; in particular, the humidity of 40% or more and 90% or less is maintained at a temperature of 30°C or more and less than 60°C, and the humidity of 70% or more and 90% or less is maintained at a temperature of 60°C or more and 90°C or less. In addition, it has been found that the water of crystallization evaporates or the organic solvent cannot be completely removed due to the decrease in humidity when the temperature and humidity ranges are outside the above ranges.

Based on the above description, it will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without changing the technical intent or essential characteristics thereof. Therefore, it should be understood that the above embodiment is not restrictive, but illustrative in all aspects. The scope of protection of the invention is determined by the appended claims and not by the description preceding them, and therefore it is provided that all changes and modifications that fall within the limits and limits of the claims, or equivalents of such limits and limits, are within the scope of the claims.

Claims (8)

1. A method for purifying a nucleic acid, comprising: a first nucleic acid crystallization step using 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°C or less and a relative humidity of 40% or more and 90% or less, wherein the nucleic acid comprises any one or more acids selected from the group consisting of guanosine-5'-monophosphate disodium salt and inosine-5'-monophosphate disodium salt. 2. A method for purifying a nucleic acid according to claim 1, wherein the nucleic acid comprises guanosine 5'-monophosphate disodium heptahydrate or inosine 5'-monophosphate disodium 7,5-hydrate. 3. A method for purifying a nucleic acid according to claim 1, wherein the hydrophilic organic solvent is any one or more solvents selected from the group consisting of methanol and ethanol. 4. The nucleic acid purification method according to 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. 5. The nucleic acid purification method according to claim 1, wherein the air has a temperature of 70°C or more and 90°C or less and a relative humidity of 70% or more and 90% or less.

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