Cyclic-di-amp sodium salt crystals

Abstract

The freeze-dried product of cyclic-di-AMP is not suitable for mass production because it needs to be manufactured using special equipment. Cyclic-di-AMP free acid crystals are not stable under harsh conditions at 105°C. Here, a cyclic-di-AMP crystal that can be easily obtained in large quantities and has excellent stability under harsh conditions at 105°C becomes a problem. The c-di-AMP sodium salt crystal of the present invention is also extremely stable under harsh conditions at 105°C. In addition, the c-di-AMP sodium salt crystal of the present invention can be mass-produced by a simple method of adjusting a c-di-AMP aqueous solution to a pH of 5.2 to 12.0 and then adding an organic solvent.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201980068123.X, filed on October 31, 2019, entitled "Cyclic-di-AMP sodium salt crystals". [Technical Field] This invention relates to a sodium salt crystal of Cyclic-di-AMP, which is considered a useful substance as an adjuvant. [Background Technology] Cyclic-di-AMP (hereinafter referred to as "c-di-AMP") is a substance discovered as a second messenger in bacteria. In recent years, it has been reported that this substance can induce type 1 interferon and holds promise for use as a drug. (Non-Patent Literature 1).

[0004] To date, methods for manufacturing c-di-AMP include chemical synthesis (Non-Patent Literature 2, 3) and enzymatic synthesis using disediic acid cyclase derived from Bacillus or Streptococcus (Non-Patent Literature 4, 5).

[0005] Currently, commercially available c-di-AMP is a lyophilized product. Regarding the acquisition of crystals, only the free acid crystals shown in Patent Document 1 have been reported (Patent Document 1). Some are sold as crystalline solids, but due to their irregular shape and tendency to crumble rather than crack when crushed, commercially available crystalline solids cannot be crystallized. Figure 1 ).

[0006] [Existing Technical Documents] [Patent Documents] [Patent Document 1] No. WO2015 / 137469 [Non-patent literature] [Non-Patent Literature 1] Science, 328, 1703-1705 (2010) [Non-Patent Literature 2] SYNTHESIS, 24, 4230-4236 (2006) [Non-Patent Literature 3] Nucleosides Nucleotides Nucleic Acids, 32, 1-16 (2013) [Non-Patent Literature 4] Molecular Cell, 30, 167-178 (2008) [Non-Patent Literature 5] Nagoya J. Med. Sci., 73, 49-57 (2011) [Summary of the Invention] [The problem the invention aims to solve] Freeze-dried products are generally known products of c-di-AMP, but the manufacturing process requires a freeze dryer, which naturally limits large-scale mass production. Therefore, there is a need to develop a crystal acquisition method that can easily obtain large quantities of crystals without the use of special equipment such as freeze dryers.

[0007] Furthermore, in the inventors' research, it was found that existing c-di-AMP free acid crystals have the disadvantage of reduced stability under harsh conditions (e.g., at 105°C).

[0008] [Solutions for solving the problem] As a result of in-depth research on the crystallization of c-di-AMP, the inventors obtained sodium salt crystals of c-di-AMP for the first time, thus completing this invention.

[0009] [The effects of the invention] In the inventors' research, it was found that c-di-AMP sodium salt crystals obtained by the following steps are extremely stable even under harsh conditions of 105°C: (1) adding alkali and / or acid to a c-di-AMP aqueous solution to adjust the pH to 5.2~12.0; (2) adjusting the absorbance OD of the c-di-AMP aqueous solution at a measurement wavelength of 257 nm. 257 The process is as follows: (1) to make 500 to 20000; (2) to heat the c-di-AMP aqueous solution to 30 to 70°C; (3) to add an organic solvent to the c-di-AMP aqueous solution; (4) to cool the c-di-AMP aqueous solution to 1 to 30°C.

[0010] Furthermore, compared with existing c-di-AMP free acid crystals, the c-di-AMP sodium salt crystals of the present invention exhibit high solubility.

[0011] In particular, the pH of step (1) is important for crystallization, and crystallization is inherently difficult under acidic conditions below pH 5.2, and even if crystals can be obtained, the yield will be low and not preferred. Conversely, sodium salt crystals are easily obtained only under conditions above pH 5.2. Therefore, the method for manufacturing c-di-AMP sodium salt crystals of the present invention is suitable for the mass production of c-di-AMP salt crystals. [Attached Image Description] Figure 1 This is a photograph showing the appearance of a commercially available c-di-AMP crystalline solid when spread out.

[0013] Figure 2 A crystal photograph showing the c-di-AMP sodium salt crystal α obtained in Example 1.

[0014] Figure 3The X-ray diffraction pattern represents the c-di-AMP sodium salt crystal α obtained in Example 1.

[0015] Figure 4 The infrared absorption spectrum of c-di-AMP sodium salt crystal α obtained in Example 1 is shown.

[0016] Figure 5 The results of thermogravimetric analysis / differential thermal analysis of c-di-AMP sodium salt crystal α obtained in Example 1 are shown.

[0017] Figure 6 A crystal photograph showing the c-di-AMP sodium salt crystal α obtained in Example 2.

[0018] Figure 7 The X-ray diffraction pattern represents the c-di-AMP sodium salt crystal α obtained in Example 2.

[0019] Figure 8 The infrared absorption spectrum of c-di-AMP sodium salt crystal α obtained in Example 2 is shown.

[0020] Figure 9 The results of thermogravimetric analysis / differential thermal analysis of c-di-AMP sodium salt crystal α obtained in Example 2 are shown.

[0021] Figure 10 This is a crystal photograph of c-di-AMP sodium salt crystal β obtained in Example 4.

[0022] Figure 11 The X-ray diffraction pattern represents the c-di-AMP sodium salt crystal β obtained in Example 4.

[0023] Figure 12 The infrared absorption spectrum of c-di-AMP sodium salt crystal β obtained in Example 4 is shown.

[0024] Figure 13 The thermogravimetric / differential thermal analysis results of c-di-AMP sodium salt crystal β obtained in Example 4 are shown.

[0025] Figure 14 A crystal photograph representing c-di-AMP free acid crystals.

[0026] Figure 15 The X-ray diffraction pattern represents the free acid crystal of c-di-AMP.

[0027] Figure 16 The infrared absorption spectrum of c-di-AMP free acid crystals is shown.

[0028] Figure 17This represents the thermogravimetric / differential thermal analysis results of c-di-AMP free acid crystals.

[0029] Figure 18 The results of stability tests under harsh conditions (105°C) are shown for the c-di-AMP free acid crystals obtained in the reference example, the sodium salt crystals α obtained in Example 1 and Example 2, and the sodium salt crystals β obtained in Example 4.

Detailed Implementation Methods

[0031]

Chemical Formula 1

[0032] When analyzed by atomic absorption spectrophotometry, the sodium content of the c-di-AMP sodium salt crystal α of the present invention is in the range of 6.2% to 6.8%. This indicates that the ratio of sodium atoms to c-di-AMP1 molecules in crystal α is 2.

[0033] The c-di-AMP sodium salt crystal α of the present invention is obtained as a columnar crystal (see reference). Figure 2 and Figure 6 ).

[0034] Furthermore, when analyzed using Cu-Kα powder X-ray diffractometer, the sodium salt crystal α of the present invention, as shown in the examples described later, exhibits characteristic peaks near diffraction angles (2θ) of 10.9 and 23.9 (°) (reference). Figure 3 and Figure 7 ).

[0035] Generally, since the diffraction angle (2θ) of powder X-ray diffraction includes an error range of less than 5%, crystals with diffraction angles of peaks that are completely uniform in powder X-ray diffraction are also included in the sodium salt crystal α of this invention, except for crystals whose peaks have uniform diffraction angles with an error of less than 5%. For example, in powder X-ray diffraction, the diffraction angle (2θ) has characteristic peaks at 10.9 ± 0.5 and 23.9 ± 1.2 (°).

[0036] When measuring the infrared absorption spectrum, the sodium salt crystal α of the present invention showed values ​​of 3118, 1604, 1221, and 1074 cm⁻¹. -1Characteristic peaks are located near (reference) Figure 4 and Figure 8 ).

[0037] It should be noted that infrared absorption spectroscopy measurements generally include areas smaller than 2 cm⁻¹. -1 The error range, except for crystals whose peak positions in the infrared absorption spectrum are exactly the same as the above values, is less than 2 cm⁻¹. -1 Crystals with consistent errors are also included in the sodium salt crystal α of this invention. For example, when measuring infrared absorption spectra, the values ​​at 3118±1.9, 1604±1.9, 1221±1.9, and 1074±1.9 (cm²) are consistent. -1 It has characteristic peaks.

[0038] When analyzed using a thermogravimetric / differential thermal analysis (TG / DTA) instrument (heating rate 5°C / min), the sodium salt crystals α of the present invention exhibit an endothermic peak near 255°C (reference). Figure 5 and Figure 9 ).

[0039] When analyzed by atomic absorption spectrophotometry, the sodium content of the c-di-AMP sodium salt crystal β of the present invention is in the range of 4.7% to 5.2%. Therefore, the ratio of sodium atoms to c-di-AMP1 molecules in crystal β is 1.5.

[0040] The c-di-AMP sodium salt crystal β of the present invention is obtained as a cubic crystal (see reference). Figure 10 ).

[0041] Furthermore, when analyzed using a powder X-ray diffractometer with Cu-Kα rays, the sodium salt crystal β of the present invention, as shown in the examples described later, exhibits characteristic peaks near diffraction angles (2θ) of 9.3, 23.6, and 24.3 (°) (reference). Figure 11 ).

[0042] Generally, since the diffraction angle (2θ) of powder X-ray diffraction includes an error range of less than 5%, crystals with peaks of powder X-ray diffraction that are completely uniform in diffraction angle are also included in the sodium salt crystal β of this invention, except for crystals whose peaks of powder X-ray diffraction have completely uniform diffraction angles. For example, the diffraction angle (2θ) in powder X-ray diffraction has characteristic peaks at 9.3±0.5, 23.6±1.2, and 24.3±1.2 (°).

[0043] When measuring the infrared absorption spectrum, the sodium salt crystal of the present invention showed β values ​​at 3119, 1606, 1222, and 1074 cm⁻¹. -1 Characteristic peaks are located near (reference) Figure 12 ).

[0044] It should be noted that infrared absorption spectroscopy measurements generally include samples smaller than 2 cm⁻¹.-1 The error range, except for crystals whose peak positions in the infrared absorption spectrum are exactly the same as the above values, is less than 2 cm⁻¹. -1 Crystals with consistent errors are also included in the sodium salt crystal β of this invention. For example, when measuring infrared absorption spectra, the values ​​at 3119±1.9, 1606±1.9, 1222±1.9, and 1074±1.9 (cm²) are consistent. -1 It has characteristic peaks.

[0045] When analyzed using a thermogravimetric / differential thermal analysis (TG / DTA) instrument (heating rate 5°C / min), the sodium salt crystals of this invention exhibit an endothermic peak around 239°C (reference). Figure 13 ).

[0046] When purity was tested using high-performance liquid chromatography, the c-di-AMP sodium salt crystals of the present invention exhibited a purity of 97% or higher, more preferably 99% or higher. Furthermore, compared to existing c-di-AMP free acid crystals, the c-di-AMP sodium salt crystals of the present invention showed high solubility.

[0047] Next, the method for preparing sodium salt crystals of c-di-AMP according to the present invention will be described. The c-di-AMP used for crystallization can be synthesized according to known methods such as enzymatic synthesis or chemical synthesis. In the synthesis, known methods can be used, for example, the methods described in Non-Patent Literature 2-5. After the reaction, the c-di-AMP generated in the reaction solution can be purified by activated carbon or reversed-phase chromatography.

[0048] The sodium salt crystals of the present invention can be obtained by adjusting the pH of the c-di-AMP aqueous solution to 5.2~12.0 and adding an organic solvent.

[0049] When the pH of the c-di-AMP aqueous solution is 5.2~6.0, the obtained sodium salt crystals are crystal β; when the pH is 6.0~12.0, the obtained sodium salt crystals are crystal α.

[0050] At pH values ​​less than 5.2, high-concentration c-di-AMP aqueous solutions will precipitate, making it difficult to obtain pure c-di-AMP sodium salt crystals. Setting the concentration of the c-di-AMP aqueous solution to a low level to avoid precipitation results in either no c-di-AMP sodium salt crystals being obtained, or only trace amounts of c-di-AMP, making efficient crystal production impossible. Consequently, to achieve large-scale and efficient c-di-AMP preparation at an industrially viable level, the pH must be set above 5.2.

[0051] In the above crystallization process, in order to obtain crystals in higher yield, it is preferable to perform the following steps: (1) adding alkali and / or acid to the c-di-AMP aqueous solution to adjust the pH to 5.2 to 12.0; (2) measuring the absorbance OD of the c-di-AMP aqueous solution at a wavelength of 257 nm. 257 The steps are: (1) setting the step size to 500~20000; (2) heating the c-di-AMP aqueous solution to 30~70°C; (3) adding an organic solvent to the c-di-AMP aqueous solution; and (4) cooling the c-di-AMP aqueous solution to 1~30°C.

[0052] In the above step (1), if the pH of the c-di-AMP aqueous solution is in the range of 5.2 to 12.0, c-di-AMP sodium salt crystals can be obtained. However, from the viewpoint that it is easy to prepare a high-concentration aqueous solution, the pH is preferably 5.4 or higher, and particularly preferably 5.6 or higher. In addition, considering the convenience of the aqueous solution being near neutral when dissolving crystals, the pH of the c-di-AMP aqueous solution is preferably 7.0 to 11.0, more preferably 7.0 to 10.0, and even more preferably 7.0 to 8.5. At this time, crystal α can be obtained by adding an organic solvent.

[0053] Examples of acids used in step (1) above include hydrochloric acid, sulfuric acid, and nitric acid, but are not limited thereto. Examples of bases used include sodium hydroxide and sodium acetate, but are not limited thereto. To prevent amorphization and rapid precipitation of crystals due to rapid addition of acid or base, it is preferable to add acid or base slowly. In the above process (2), the absorbance OD of the c-di-AMP aqueous solution at a measurement wavelength of 257 nm was measured. 257 When the concentration is 500 or higher, crystals can be obtained by adding an organic solvent. However, considering the need to reduce the amount of organic solvent required for crystal precipitation, it is preferable to add 1000 or higher, more preferably 2000 or higher, and even more preferably 3000 or higher. Furthermore, when the c-di-AMP aqueous solution is at a high concentration, considering the increased viscosity and reduced operability, the absorbance OD of the c-di-AMP aqueous solution at a measurement wavelength of 257 nm is... 257 Preferably, it is below 20,000, more preferably below 15,000, and even more preferably below 10,000.

[0054] In step (3) above, the temperature of the c-di-AMP aqueous solution is heated to 30~70°C. Since there is a temperature difference between step (5) and cooling, crystals are more likely to precipitate. Therefore, the temperature of the aqueous solution in step (3) is preferably 40°C or higher, and more preferably 50°C or higher.

[0055] The c-di-AMP aqueous solution used in this crystal acquisition process may contain organic solvent within a range that prevents crystal precipitation. However, from the viewpoint of preventing accidental crystal precipitation, the content of organic solvent is preferably 30% (v / v) or less, more preferably 20% (v / v) or less, more preferably 10% (v / v) or less, more preferably 5% (v / v) or less, and more preferably substantially free of organic solvent.

[0056] Examples of organic solvents used in step (4) above include alcohols with 6 or fewer carbon atoms, such as methanol and ethanol; ketones, such as acetone; ethers, such as dioxane; nitriles, such as acetonitrile; and amides, such as dimethylformamide, but are not limited thereto. In particular, from the viewpoint of ease of use and safety, alcohols with 6 or fewer carbon atoms are preferred, with ethanol being the most preferred.

[0057] In step (5) above, the c-di-AMP aqueous solution is cooled to 1~30°C. Since there is a temperature difference between step (3) and heating, crystals are more likely to precipitate. Therefore, the temperature of the aqueous solution in step (5) is preferably below 20°C, and more preferably below 10°C.

[0058] In addition, it is preferable to perform the above steps (1) to (5) in sequence, but it is appropriate to perform consecutive steps simultaneously.

[0059] The c-di-AMP sodium salt crystals obtained by the above manufacturing method can be dried after filtration and extraction to obtain the product. During drying, methods such as vacuum drying can be appropriately utilized.

[0060]

Example

[0061] (Example 1) Preparation of c-di-AMP sodium salt crystals α at pH 8.2 c-di-AMP was synthesized enzymatically using a known method and then purified.

[0062] The purified OD 257 A 102 mL solution of c-di-AMP at pH 8.2 (4710) was heated to 40 °C. Then, 142 mL of 99.5% (w / w) ethanol was slowly added while stirring, and the liquid temperature was cooled to 4 °C to precipitate crystals. The precipitated crystals were filtered using a basket separator to obtain wet crystals. The wet crystals were dried at 30 °C for 2 hours to obtain 9.8 g of dried crystals.

[0063] (A) Speciation analysis of sodium salt Atomic absorption spectrophotometry analysis revealed that the sodium content in the c-di-AMP sodium salt crystal of this embodiment was 6.4%. Therefore, the ratio of sodium atoms to c-di-AMP1 molecules was 2.0, and the c-di-AMP sodium salt crystal α of this embodiment exhibited a 2-sodium salt morphology.

[0064] (B) Purity Test The purity of the c-di-AMP sodium salt crystals α obtained in Example 1 was analyzed by high performance liquid chromatography (HPLC), and the purity of c-di-AMP was 100%. It should be noted that the HPLC was performed under the following conditions.

[0065] (condition) Column: Hydrosphere C18 (made by YMC Corporation) Eluent: 0.1M TEA-P (pH 6.0) + 4.5% ACN Detection method: UV 260 nm detection (C) Crystal form A photograph of c-di-AMP sodium salt crystal α in this embodiment is shown in Figure 2 .like Figure 2 As shown, c-di-AMP sodium salt crystal α is clearly columnar in shape.

[0066] (D) Powder X-ray diffraction For the c-di-AMP sodium salt crystal α in this embodiment, the X-ray diffraction pattern was measured using an X'Pert PRO MPD (Speedy Corporation) under the following measurement conditions.

[0067] (Measurement conditions) Object: Cu X-ray tube current: 40mA X-ray tube voltage: 45kV Scanning range: 2θ = 4.0~40.0° Pretreatment: Grind using an agate mortar and pestle. like Figure 3 As shown in Table 1, the c-di-AMP sodium salt crystal α of this embodiment shows peaks near diffraction angles (2θ) of 6.2, 10.9, 12.6, and 23.9 (°), especially characteristic peaks near 10.9 and 23.9 (°).

[0068] Table 1 (E) Infrared absorption spectrum For the c-di-AMP sodium salt crystal α in this embodiment, the infrared absorption spectrum was measured using a Fourier transform infrared spectrophotometer SpectrumOne (Perkin Elmer) via ATR (Attenuated Total Reflectance).

[0069] In this embodiment, the c-di-AMP sodium salt crystals α were at 3119, 1604, 1220, and 1073 (cm²). -1 Characteristic peaks are found near the ) area. These results are shown in Figure 4 .

[0070] (F) Differential scanning thermal analysis Analysis using a thermogravimetric / differential thermal analysis (TG / DTA) instrument (heating rate 5°C / min) revealed that the c-di-AMP sodium salt crystals in this embodiment exhibit an endothermic peak around 255°C. Figure 5 ).

[0071] (Example 2) Preparation of c-di-AMP sodium salt crystals α at pH 10.0 c-di-AMP was synthesized enzymatically using a known method and then purified.

[0072] The purified OD 257 A 50 mL solution of c-di-AMP at pH 10.0 (6600) was heated to 40 °C. Then, 45 mL of ethanol was slowly added while stirring, and the liquid temperature was cooled to 4 °C to precipitate crystals. The precipitated crystals were filtered through a 3 μm membrane filter to obtain wet crystals. The wet crystals were dried at 20 °C for 1.5 hours to obtain 7 g of dried crystals.

[0073] (A) Speciation analysis of sodium salt Atomic absorption spectrophotometry analysis revealed that the sodium content in the c-di-AMP sodium salt crystal α of this embodiment was 6.6%. Therefore, the ratio of sodium atoms to c-di-AMP1 molecules was 2.0, and the c-di-AMP sodium salt crystal α of this embodiment exhibited a 2-sodium salt morphology.

[0074] (B) Purity Test Analysis of the purity of c-di-AMP sodium salt crystals α obtained in Example 2 using high-performance liquid chromatography (HPLC) revealed that the purity of c-di-AMP was 100%. It should be noted that the HPLC was performed under the following conditions.

[0075] (condition) Column: Hydrosphere C18 (made by YMC Corporation) Eluent: 0.1M TEA-P (pH 6.0) + 4.5% ACN Detection method: UV 260 nm detection (C) Crystal form A photograph of c-di-AMP sodium salt crystal α in this embodiment is shown in Figure 6 .like Figure 6 As shown, c-di-AMP sodium salt crystal α is clearly columnar in shape.

[0076] (D) Powder X-ray diffraction For the c-di-AMP sodium salt crystal α in this embodiment, the X-ray diffraction pattern was measured using an X'Pert PRO MPD (Speedy Corporation) under the following measurement conditions.

[0077] (Measurement conditions) Object: Cu X-ray tube current: 40mA X-ray tube voltage: 45kV Scanning range: 2θ = 4.0~40.0° Pretreatment: Grind using an agate mortar and pestle. like Figure 7 As shown in Table 2, the c-di-AMP sodium salt crystal α in this embodiment exhibits characteristic peaks near diffraction angles (2θ) of 10.9 and 24.0 (°).

[0078] Table 2 (E) Infrared absorption spectrum For the c-di-AMP sodium salt crystal α in this embodiment, the infrared absorption spectrum was measured using a Fourier transform infrared spectrophotometer SpectrumOne (Perkin Elmer) via the ATR (Attenuated Total Reflectance) method.

[0079] In this embodiment, the c-di-AMP sodium salt crystals α were at 3118, 1604, 1221, and 1074 cm⁻¹. -1 Characteristic peaks are found near the ) area. These results are shown in Figure 8 .

[0080] (F) Differential scanning thermal analysis Analysis using a thermogravimetric / differential thermal analysis (TG / DTA) instrument (heating rate 5°C / min) revealed that the c-di-AMP sodium salt crystals in this embodiment exhibit an endothermic peak around 255°C. Figure 9 ).

[0081] (Example 3) Experiment on the acquisition of c-di-AMP sodium salt crystals in a low pH range (pH 5.0~6.5) To determine the pH range in which c-di-AMP sodium salt crystals could be obtained, crystal acquisition experiments were conducted in a low pH range (pH 5.0–6.5).

[0082] c-di-AMP was synthesized enzymatically using a known method and then purified.

[0083] OD was prepared using purified c-di-AMP. 257 An aqueous solution of c-di-AMP at 2500 oz and pH values ​​of 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, or 6.5 was obtained. The resulting OD... 257 A 2500 mL c-di-AMP solution (0.2 mL) was heated to 30 °C. 1 mL of 99.5% (w / w) ethanol was slowly added, the solution was sealed, and the mixture was left at 30 °C overnight to several days to precipitate crystals. The precipitated crystals were then extracted by filtration through a membrane filter to obtain wet crystals.

[0084] The results are shown in Table 3 below. In Table 3, "crystal" indicates whether crystals can be obtained; other symbols are as follows: ○: Able to obtain crystals without any problems △: Crystals can be obtained, but in much smaller quantities compared to other pH values.

[0085] ×: It is impossible to prepare an aqueous solution or obtain crystals.

[0086] Table 3 As shown in Table 3 above, the solubility of c-di-AMP is low at pH 5.0, making it impossible to prepare OD. 257 The solution was a 2500 mL c-di-AMP aqueous solution. Low-concentration c-di-AMP aqueous solutions were prepared by dilution, but subsequent identical operations failed to yield crystals. Crystals were obtained at pH 5.2, but the amount precipitated was less than at pH 5.4 and above. Crystals were obtained without any problems at pH 5.4 and above.

[0087] C-di-AMP crystals obtained at pH values ​​above 5.4 and below 6.0 exhibited the same cubic appearance and other properties, and were considered to have the same morphology. At pH 6.0, both cubic and columnar crystals were observed. C-di-AMP crystals obtained at pH values ​​above 6.0 exhibited columnar appearance and other properties, showing the same characteristics as the crystals obtained in Examples 1 and 2. These results indicate that c-di-AMP crystal β of the present example was obtained when the pH of the c-di-AMP aqueous solution was in the range of 5.2 to 6.0, and c-di-AMP sodium salt crystal α was obtained when the pH was in the range of 6.0 to 12.0.

[0088] (Example 4) Preparation of c-di-AMP sodium salt crystals β at pH 5.6 c-di-AMP was synthesized enzymatically using a known method and then purified.

[0089] The purified OD 257 A 15 mL solution of c-di-AMP at pH 5.6 (3000) was heated to 30 °C. Then, 15 mL of ethanol was slowly added while stirring, and the liquid temperature was cooled to 4 °C to precipitate crystals. The precipitated crystals were filtered through a membrane filter to obtain wet crystals. The wet crystals were dried at 25 °C for 2 hours to obtain 1.0 g of dried crystals.

[0090] (A) Speciation analysis of sodium salt Atomic absorption spectrophotometry analysis revealed that the sodium content in the c-di-AMP sodium salt crystal β of this embodiment was 5.2%. Therefore, the ratio of sodium atoms to c-di-AMP1 molecules was 1.5.

[0091] (B) Purity Test Analysis of the purity of c-di-AMP sodium salt crystals β obtained in this example by high performance liquid chromatography (HPLC) revealed that the purity of c-di-AMP was 100%. It should be noted that the HPLC was performed under the following conditions.

[0092] (condition) Pillar: YMC-Triart C18 (Made by YMC Corporation) Eluent: 0.1M TEA-P (pH 6.0) + 4.5% ACN Detection method: UV 260 nm detection (C) Crystal form A photograph of c-di-AMP sodium salt crystal β in this embodiment is shown in Figure 10 .like Figure 10 As shown, the c-di-AMP sodium salt crystal β is clearly cubic in shape.

[0093] (D) Powder X-ray diffraction For the c-di-AMP sodium salt crystal of this embodiment, the X-ray diffraction pattern was measured using an X'Pert PRO MPD X-ray diffractometer under the following measurement conditions.

[0094] (Measurement conditions) Object: Cu X-ray tube current: 40mA X-ray tube voltage: 45kV Scanning range: 2θ = 4.0~40.0° Pretreatment: Grind using an agate mortar and pestle. like Figure 11 As shown in Table 4, the c-di-AMP sodium salt crystal β in this embodiment exhibits peaks near diffraction angles (2θ) of 9.3, 23.6, and 24.3 (°).

[0095] Table 4 (E) Infrared absorption spectrum For the c-di-AMP sodium salt crystal β in this embodiment, the infrared absorption spectrum was measured using a Fourier transform infrared spectrophotometer SpectrumOne (Perkin Elmer) via the ATR (Attenuated Total Reflectance) method.

[0096] In this embodiment, the c-di-AMP sodium salt crystals β values ​​were 3119, 1606, 1222, and 1074 (cm²). -1 Characteristic peaks are found near the ) area. These results are shown in Figure 12 .

[0097] (F) Differential scanning thermal analysis Analysis using a thermogravimetric / differential thermal analysis (TG / DTA) instrument (heating rate 5°C / min) revealed that the c-di-AMP sodium salt crystals in this embodiment exhibit an endothermic peak around 239°C. Figure 13 ).

[0098] (Example for reference) Fabrication of c-di-AMP free acid crystals c-di-AMP was synthesized enzymatically using a known method and then purified.

[0099] c-di-AMP free acid crystals were obtained from the purified c-di-AMP solution according to the method described in Patent Document 1. That is, OD... 257 A 114-c-di-AMP solution (980 mL) was heated to 50 °C. 2N HCl was then added dropwise until the pH reached 1.8. The solution was cooled to 4 °C, and crystals precipitated. The precipitated crystals were filtered through a glass filter (G3) to obtain wet crystals. The wet crystals were dried at 20 °C for 1 hour to obtain 2.8 g of dried crystals.

[0100] (A) Purity test The purity of the c-di-AMP free acid crystals obtained in this example was determined by high performance liquid chromatography (HPLC), and the purity of c-di-AMP was 100%. It should be noted that the HPLC was performed under the following conditions.

[0101] (condition) Column: Hydrosphere C18 (made by YMC Corporation) Eluent: 0.1M TEA-P (pH 6.0) + 4.5% ACN Detection method: UV 260 nm detection (B) Crystal form A representative photograph of c-di-AMP free acid crystals is shown in Figure 14 c-di-AMP free acid crystals exhibit a needle-like crystal form.

[0102] (C) Powder X-ray diffraction For c-di-AMP free acid crystals, X-ray diffraction patterns were determined using an X'Pert PRO MPD X-ray diffractometer under the following conditions.

[0103] (Measurement conditions) Object: Cu X-ray tube current: 40mA X-ray tube voltage: 45kV Scanning range: 2θ = 4.0~40.0° Pretreatment: Grind using an agate mortar and pestle. like Figure 15 As shown in Table 5, the c-di-AMP free acid crystal described in Patent Document 1 exhibits characteristic peaks near diffraction angles (2θ) of 9.2, 10.2, 10.9, 11.1, 13.7, 15.2, 19.0, 20.6, 22.4, 23.1, 24.3, 26.6, and 26.8 (°).

[0104] Table 5 (D) Infrared absorption spectrum The results of measuring the infrared absorption spectrum of c-di-AMP free acid crystals using a Fourier transform infrared spectrophotometer Spectrum One (Perkin Elmer) via the ATR (Attenuated Total Reflectance) method are described in Patent Document 1.

[0105] The c-di-AMP free acid crystals described in Patent Document 1 have values ​​of 3087, 1686, 1604, 1504, 1473, 1415, 1328, and 1213 cm⁻¹. -1 Characteristic peaks are found near the ) area. These results are shown in Figure 16 .

[0106] (E) Differential scanning thermal analysis Analysis of the c-di-AMP free acid crystals described in Patent Document 1 using a thermogravimetric / differential thermal analysis (TG / DTA) instrument (heating rate 5℃ / min) revealed an endothermic peak near 193℃. Figure 17 ).

[0107] (Example 5) Stability test of crystals obtained at 105°C The crystals obtained in Examples 1, 2, 4, and the Reference Example were left to stand at 105°C. Over time, the crystals were recovered to prepare an aqueous solution, and the purity of the crystals was analyzed by high-performance liquid chromatography (HPLC). The results are shown below. Figure 18 And Table 6.

[0108] Table 6 As shown in Table 6, compared to existing c-di-AMP free acid crystals, the c-di-AMP sodium salt crystals of this invention exhibit superior stability under harsh conditions of 105°C. Furthermore, unlike existing c-di-AMP free acid crystals which require stirring to dissolve, the c-di-AMP sodium salt crystals of this invention dissolve rapidly. Therefore, it is evident that the c-di-AMP sodium salt crystals of this invention demonstrate significantly higher solubility compared to existing c-di-AMP free acid crystals.

Claims

1. A method for manufacturing Cyclic-di-AMP sodium salt crystals, characterized in that, The crystal is either Cyclic-di-AMP sodium salt crystal α or Cyclic-di-AMP sodium salt crystal β. The Cyclic-di-AMP sodium salt crystal α is characterized by having characteristic peaks at 10.9±0.5 and 23.9±1.2 (°) as the diffraction angle (2θ) of powder X-ray diffraction using Cu-Kα rays. The Cyclic-di-AMP sodium salt crystal β is characterized by having characteristic peaks at 9.3±0.5, 23.6±1.2, and 24.3±1.2 (°) as the diffraction angle (2θ) of powder X-ray diffraction using Cu-Kα rays. The manufacturing method includes: The process of adjusting the pH of the Cyclic-di-AMP aqueous solution to 5.2–6.0 and then adding an organic solvent to obtain the precipitated Cyclic-di-AMP sodium salt crystals β; or The process involves adjusting the pH of the Cyclic-di-AMP aqueous solution to 6.0–12.0 and then adding an organic solvent to obtain the precipitated Cyclic-di-AMP sodium salt crystals α.

2. The method for manufacturing Cyclic-di-AMP sodium salt crystals according to claim 1, characterized in that, The process includes the following steps: (1) The step of adding alkali and / or acid to the c-di-AMP aqueous solution to adjust the pH to 5.2-6.0 or 6.0-12.0; (2) The process of making the absorbance OD257 of the c-di-AMP aqueous solution at a measurement wavelength of 257nm reach 500 to 20000; (3) The process of heating the c-di-AMP aqueous solution to 30-70°C; (4) The process of adding organic solvent to the c-di-AMP aqueous solution; (5) The process of cooling the c-di-AMP aqueous solution to 1 to 30°C.

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