Method for purifying peptide and peptide purified using same

Abstract

The present invention provides: a method for purifying a salt form of a peptide, the method comprising (a) a crude peptide preparation step, (b) a salt compound formation step, and (c) a salt compound separation step; and a salt form of a peptide purified by the purification method according to the present invention. The present invention has an advantage in that a large amount of peptides can be easily purified at high yield / high purity without using a chromatography method. In addition, the present invention has the advantage of being able to provide a peptide purified at high yield / high purity.

Description

Peptide purification method and peptide purified using the same

[0001] The present invention relates to a peptide purification method and a peptide purified using the same, and more particularly, to a peptide purification method capable of easily purifying a salt-form peptide with high yield / high purity without using a chromatography method, and to a peptide in salt form purified using the same.

[0002] Methods for chemically synthesizing peptides can be broadly divided into liquid-phase synthesis and solid-phase synthesis. Liquid-phase peptide synthesis, a classical synthesis method, suffers from the difficulty of isolating and purifying the target product. Furthermore, solid-phase peptide synthesis, developed by R.B. Merrifield in 1963, has the advantage of being automatable and is widely used. However, this method also requires purification to obtain the target product with the desired purity. Chromatography has been used for this purification (see, e.g., International Patent Application Publication No. WO 2009 / 098707 A1). However, purifying peptides using chromatographic methods (e.g., reversed-phase liquid chromatography, reversed-phase high-performance liquid chromatography) has the disadvantage of low yields. For example, when purifying peptides by reversed-phase liquid chromatography or reversed-phase high-performance liquid chromatography, the yield usually does not even reach 50%, and due to the nature of the chromatography method, the amount of purification per time can only be small, so there are problems in terms of economy and productivity. In addition, there are problems such as high cost, low productivity, and burden on the environment due to the fact that a large amount of purified water and solvent must be used, and since the freeze-drying method is mainly used to obtain the final target product, a lot of time and energy are required in addition to expensive freeze-drying equipment, and expensive chromatography equipment must be maintained.

[0003] Therefore, there is a need to develop technologies that can solve such problems.

[0004] [Prior Art Literature]

[0005] [Patent Document]

[0006] (Patent Document 1) International Patent Application Publication No. WO 2009 / 098707 A1, August 13, 2009, Specification

[0007] One problem that the present invention seeks to solve is to provide a peptide purification method capable of purifying a peptide with high yield and high purity without using chromatography.

[0008] In addition, another problem that the present invention seeks to solve is to provide a peptide purified with high yield and high purity without using chromatography.

[0009] The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0010] The present invention provides a method for purifying a peptide in salt form, comprising: (A) a crude peptide preparation step of preparing a crude peptide in salt form; (B) a salt compound formation step of adding the crude peptide in salt form to a solvent and adding an acid to form a salt compound; and (C) a salt compound separation step of separating the salt compound from the solvent.

[0011] In addition, the present invention provides a peptide in salt form purified by the purification method of the present invention.

[0012] The present invention has the effect of enabling the easy purification of large quantities of peptides with high yield and high purity, without resorting to chromatography. Furthermore, the present invention has the effect of providing peptides purified with high yield and high purity.

[0013] Figure 1 is a flowchart for explaining one embodiment of the present invention.

[0014] Hereinafter, the present invention will be described in more detail by examples and manufacturing examples. However, the following examples and manufacturing examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples or manufacturing examples.

[0015] In the present invention, a peptide is a polymer in which multiple amino acid units are covalently linked by peptide bonds. Preferably, the peptide refers to a polymer of a length that allows for chemical synthesis. The peptide of the present invention may be, but is not limited to, a peptide of up to 50 amino acids in length, as long as chemical synthesis is possible.

[0016] A method for purifying a peptide in a salt form, which is one embodiment of the present invention, is described with reference to FIG. 1. FIG. 1 is a flowchart illustrating one embodiment of the present invention. As illustrated in FIG. 1, the purification method, which is one embodiment of the present invention, includes (A) a crude peptide preparation step; (B) a salt compound formation step; and (C) a salt compound separation step.

[0017] (A) The crude peptide preparation step is the step of preparing the crude peptide in salt form. The crude peptide refers to the peptide to be purified before purification (e.g., purity of 90% or less).

[0018] A crude peptide in salt form can exhibit acidity. That is, a crude peptide in salt form can be a peptide that exhibits acidity (e.g., pH below 7). For example, a peptide salt formed from an acid can act as an acid in a solvent because at least the C-terminal carboxyl group is free. Therefore, an example of a crude peptide in salt form is an acid addition salt, and a peptide with a free C-terminal carboxyl group can be used.

[0019] Specifically, the crude peptide in salt form may be synthesized by a solid-phase peptide synthesis method. The peptide synthesized by the solid-phase peptide synthesis method may be a salt form of trifluoroacetic acid. Therefore, the salt form may be a salt form of trifluoroacetic acid. Specifically, the crude peptide in salt form may be a crude peptide in the salt form of trifluoroacetic acid. By directly purifying the crude peptide in salt form synthesized by the solid-phase peptide synthesis method, purification can be performed more simply.

[0020] (B) The salt compound formation step is a step in which the salt form of the crude peptide is added to a solvent and an acid is added to form a salt compound. In this case, the "salt compound" may refer to a salt form of the peptide formed by further adding an acid to the salt form of the crude peptide. By adding an acid to the salt form of the crude peptide to form a salt compound, the crude peptide can be induced to become a more complete salt state, and as a result, the peptide appears to be purified more effectively.

[0021] Here, the solvent is not limited as long as it can dissolve the salt form of the crude peptide, and may be a single solvent or a mixed solvent. The single solvent may be, for example, acetone, methanol, ethanol, propanol, or butanol. Propanol includes n-propanol and / or isopropanol. In addition, butanol includes n-butanol and / or tert-butanol.

[0022] A mixed solvent can be formed by mixing two or more single solvents or by adding an auxiliary solvent to one or more single solvents. That is, a mixed solvent can be formed by mixing two or more single solvents, or by adding an auxiliary solvent to a single solvent. It appears that the auxiliary solvent mainly promotes the formation of solid-phase crystals of salt compounds, and thus the separation of salt compounds is easier and shows a good yield. Such an auxiliary solvent is not limited as long as it can promote the separation of salt compounds, but may be, for example, one or more selected from ethyl acetate or ethyl ether. A mixed solvent with such an auxiliary solvent added may be, for example, a mixture of one or more selected from acetone, methanol, ethanol, propanol, or butanol and one or more selected from ethyl acetate or ethyl ether.

[0023] Among the mixed solvents, the auxiliary solvent may preferably be present in an amount of 1 to 50 vol%, more preferably 5 to 40 vol%, and even more preferably 10 to 40 vol%. Within this range, purification is possible more effectively in terms of purity and yield.

[0024] By applying such solvents, the peptides appear to be purified more effectively.

[0025] The solvent may be preferably 100 to 3000 parts by weight, more preferably 200 to 2000 parts by weight, and even more preferably 500 to 1500 parts by weight, based on 100 parts by weight of the salt form of the crude peptide. Below this range, there is a concern that the purity may decrease, and above this range, there is a concern that the yield may decrease.

[0026] By applying a solvent of this content, the peptide appears to be purified more effectively.

[0027] In addition, the formation of the salt compound can be preferably carried out at a temperature of -10°C to 70°C, more preferably at a temperature of 5°C to 70°C, and even more preferably at a temperature of 10°C to 50°C. Below this range, there is a concern that the purity may decrease, and above this range, there is a concern that the yield may decrease.

[0028] In addition, the acid may be preferably 0.5 to 3 equivalents, and more preferably 1 equivalent, relative to 1 equivalent of the salt form of the crude peptide. If it is less than this range, there is a concern that purification may be insufficient, and if it is more than this range, there is a concern that impurities may be generated and the purity may be lowered.

[0029] Additionally, the salt form of the crude peptide may be a salt form corresponding to a salt of an acid. For example, the salt form of the crude peptide may be a salt form of trifluoroacetic acid, and the acid may be trifluoroacetic acid.

[0030] Specifically, the acid is trifluoroacetic acid, and step (B) may be performed by adding trifluoroacetic acid to a solvent containing a salt form of the crude peptide, mixing at room temperature, heating to 40°C to 50°C, remixing, and then cooling to room temperature. At this time, the mixing may be performed for 0.5 hours. Additionally, the remixing may be performed for 1 hour.

[0031] Additionally, step (B) may further include a final mixing process after the cooling. In this case, the final mixing may be performed for 1 hour.

[0032] (C) The salt compound separation step is a step of separating the salt compound from the solvent. Specifically, the separation may include a process of washing and drying the filtered salt compound. At this time, the washing may be performed with the same solvent as the solvent in step (B). Filtration may be performed using a filter typically used for filtration.

[0033] Each of these steps can be performed in a standard reactor, rather than using a chromatography device. Therefore, the present invention avoids the problems associated with chromatography, and enables the easy purification of large quantities of peptides with high yield and purity.

[0034] The purification method, which is one embodiment of the present invention, can be performed in a reactor other than a chromatographic reactor. Specifically, step (B) can be performed in a reactor other than a chromatographic reactor. In this case, the reactor may be a non-chromatographic reactor. Such a reactor may not include a chromatographic stationary phase.

[0035] In addition, a purified salt form of a peptide, which is an embodiment of the present invention, can be produced by a purification method, which is an embodiment of the present invention.

[0036] The purification method and peptide, which are one embodiment of the present invention, will be examined in more detail through the following manufacturing examples.

[0037] Below, the reagents and materials used in the manufacturing examples are commercially available, and the best quality was used. Unless otherwise stated, those purchased from Sigma-Aldrich were used.

[0038] <Manufacturing Example> Manufacturing of purified peptides

[0039] 1. Preparation of crude peptide

[0040] The crude peptides in the trifluoroacetate form described in Table 1 were prepared by a general solid-phase peptide preparation method. Case 1-1 is exemplified as follows. Specifically, the starting material H-Asp(OtBu)-2-ClTrt solid resin (substitution ratio: 0.63 to 0.67 mmol / g) was used to prepare the crude peptides by a standard fluorenylmethyloxycarbonyl solid-phase peptide synthesis (Fmoc-SPPS) method. First, 125.0 g of H-Asp(OtBu)-2-ClTrt peptide resin as a starting material was added to a well-dried reactor, and N,N-dimethylformamide (DMF, 1250 ml, 10 mL / g resin) was added, stirring was performed for 20 minutes to swell the resin, and then the filtered solution was discarded and the resin was washed twice with N,N-dimethylformamide (DMF, 750 ml, 6 mL / g resin). In another reactor, Fmoc-Leu-OH (1.5 equivalents based on the starting material) and hydroxybenzotriazole (HOBt, 1.5 equivalents based on the starting material) were charged, dissolved in N,N-dimethylformamide (DMF, 4.5 mL per gram of starting material), and then diisopropylethylamine (DIPEA, 1.5 equivalents based on the starting material) was added. The reaction solution was cooled to 0-5°C, and then 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazole-1-oxide hexafluorophosphate (HBTU, 1.5 equivalents based on the starting material) was added to activate the carboxyl group of the amino acid. Dichloromethane (1.5 mL per 1 g of starting material) was added to the reactor containing the starting material peptide resin, and while stirring, a solution of an activated amino acid (Fmoc-Leu-OH) derivative prepared in another reactor was slowly added, and the reaction solution was stirred at a temperature of 10-30°C for 2 hours. The reaction was confirmed using the ninhydrin (Kaiser) test method.After the reaction was completed, the peptide-resin was filtered and washed with the solvents N,N-dimethylformamide (DMF) and methyl tert-butyl ether (MTBE), respectively, and then the peptide-resin solid was washed twice with N,N-dimethylformamide (DMF) (750 ml, 6 mL / g resin). The following fluorenylmethyloxycarbonyl (Fmoc) deprotection reaction was performed by treating the peptide-resin with a 5% piperidine mixture solution (v / v / w / v: 5% piperidine / 1.25% DBU / 1% HOBt / DMF) twice (750 mL, 6 mL / g resin, each stirred for 10 minutes and then filtered). The deprotection reaction was then performed by filtering, and the filtered peptide-resin was washed with the solvent, N,N-dimethylformamide (DMF) x 2 times, methyl t-butyl ether (MTBE) x 2 times, and N,N-dimethylformamide (DMF) x 2 times (60 mL / g resin for each solvent). The fluorenylmethyloxycarbonyl (Fmoc) deprotection reaction was confirmed by the ninhydrin (Kaiser) test. Coupling and fluorenylmethyloxycarbonyl (Fmoc) deprotection reactions for amino acid derivatives #3, #4, and #5 were performed in the same manner as above, that is, amino acids were sequentially reacted with the peptide-resin using #3 reaction Fmoc-His(Trt)-OH, #4 reaction Fmoc-Leu-OH, and #5 reaction Fmoc-Glu(OtBu)-OH to synthesize the Fmoc-Glu(OtBu)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide-resin. When amino acid #3 introduced Fmoc-His(Trt)-OH, the coupling reaction was performed using a mixed solvent, N,N-dimethylformamide / dichloromethane (DCM / DMF) and N-hydroxybenzotriazole / N,N,N-diisopropylcarbodiimide for carboxyl group activation. The N-terminal fluorenylmethyloxycarbonyl (Fmoc)-group of the Fmoc-Glu(OtBu)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide-resin was induced by using a 5% piperidine mixed solvent (v / v / w / v: 5% piperidine / 1.H-Glu(OtBu)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide-resin was synthesized by adding 25% DBU / 1% HOBt / DMF, stirring, filtering, and washing.

[0041] In another reactor, a mixed solution of trifluoroacetic acid / triisopropylsilane / dichloromethane (TFA / TIS / DCM: 55 / 5 / 40, v / v / v, 450 mL, 10 mL per 1 g of peptide resin) was added and cooled to 0-5°C. The obtained peptide-resin was slowly added to the cooled solution, while maintaining the reaction solution temperature below 17°C (optimum temperature, 13.8-14.6°C). Subsequently, the reaction solution was heated to 23-27°C and stirred at that temperature for 90 minutes. The reaction solution was filtered using a polytetrafluoroethylene (PTFE) filter to obtain a solid resin, washed twice with trifluoroacetic acid (TFA) (1.0 mL x 2 per gram of peptide resin), and the filtrates were combined. The combined filtrates were concentrated under reduced pressure at 30°C or lower until the volume became 20-30%. To the concentrated residue, pre-cooled methyl t-butyl ether (MTBE) solvent (approximately 10 times the concentrated volume) was added to produce a crude peptide as a solid precipitate. The resulting solid precipitate was stirred at room temperature for 1 hour, filtered, and washed five times with cooled methyl t-butyl ether (MTBE) solvent (50% of the amount of MTBE used for precipitation). The filtered crude peptide was vacuum-dried at 24°C for approximately 30 hours to synthesize a crude peptide (trifluoroacetate form, 57.4 g) having the amino acid sequence of SEQ ID NO: 1 with a deprotected peptide yield of 98.4% and an HPLC purity of 86%. The remaining peptides, except 1-1 described in Table 1, were also synthesized using the same solid-phase peptide preparation method as 1-1 (see WO2011 / 019123 A1, US2019 / 0161517 A1, WO2020 / 184901 A1). The synthesized crude peptides had one of amino acids 5 to 9, as described in Table 1. The purity was confirmed by HPLC, and the amino acid sequence was confirmed by LC / MS / MS. In addition, the molecular weight was confirmed by LC / MS. The results are shown in Table 1 below.

[0042] Amino acid sequence purity (%) of crude peptide Mass spectrometry Theoretical value Analysis value 1-1 Glu-Leu-His-Leu-Asp (SEQ ID NO: 1) 86625.7625.41 - 2 Leu-Gln-Val-Val-Tyr-Leu-His (SEQ ID NO: 2) 89871.0871.11 - 3 Asp-Gly-Glu-Phe-Phe (SEQ ID NO: 3) 90613.6613.61 - 4 Tyr-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr (SEQ ID NO: 4) 85785.8785.1

[0043] 2. Formation and separation of chloride compounds

[0044] 50 g of the crude peptide (trifluoroacetate form, purity 86%) consisting of the amino acid sequence of SEQ ID NO: 1 prepared previously was added to 500 mL of the solvent described in Table 2 {in a non-chromatographic reactor (e.g., batch reactor)}, 1 equivalent of trifluoroacetic acid (TFA) was slowly added, and the mixture was stirred at room temperature (25°C) for 0.5 h. The temperature was slowly increased to 40-50°C and stirred for 1 h. The reaction solution was cooled to room temperature (25°C) and stirred for 1 h. Thereafter, the mixture was filtered through a filter paper, washed with 100 ml of the solvent described in Table 2, and vacuum-dried at 30°C for about 24 h. As a result, the peptide (white or off-white) in the trifluoroacetate form was obtained in the yield and purity described in Table 2. The purity was confirmed by HPLC method.

[0045] Solvent yield purity Methanol 65% 99.2% Methanol / ethyl ether (400 / 100, v / v) 75% 97.7% Methanol / ethyl acetate (300 / 200, v / v) 74% 98.3% Ethanol 68% 98.9% Ethanol / ethyl ether (450 / 50, v / v) 72% 97.8% Isopropanol 67% 95.2% Isopropanol / ethyl ether (475 / 25, v / v) 75% 95.8% t-butanol 73% 96.9% t-butanol / ethyl ether (475 / 25, v / v) 79% 96.7% Acetone 72% 99.3%

[0046] In addition, the crude peptide (trifluoroacetate form, purity 89%) consisting of the amino acid sequence of SEQ ID NO: 2 prepared previously was subjected to the same process as the trifluoroacetate form of the crude peptide consisting of the amino acid sequence of SEQ ID NO: 1, except that the solvent described in Table 3 was used instead of the solvent described in Table 2. As a result, a peptide (white or off-white) in the form of trifluoroacetate having the yield and purity described in Table 3 was obtained. The purity was confirmed by HPLC.

[0047] Solvent yield purity Methanol 81% 99.2% Methanol / ethyl ether (400 / 100, v / v) 86% 98.4% Methanol / ethyl acetate (300 / 200, v / v) 86% 98.7% Ethanol 83% 98.9% Ethanol / ethyl ether (450 / 50, v / v) 85% 98.5% Isopropanol 88% 97.5% Isopropanol / ethyl ether (475 / 25, v / v) 90% 97.1% Acetone 85% 99.4%

[0048] In addition, the crude peptide (trifluoroacetate form, purity 90%) consisting of the amino acid sequence of SEQ ID NO: 3 prepared previously was subjected to the same process as the trifluoroacetate of the crude peptide having the amino acid sequence of SEQ ID NO: 1, except that the solvent described in Table 4 was used instead of the solvent described in Table 2. As a result, the peptide (white or off-white) in trifluoroacetate form was obtained with the yield and purity described in Table 4. The purity was confirmed by HPLC.

[0049] Solvent yield purity Methanol 90% 98.9% Ethanol 93% 98.5% Isopropanol 95% 97.8% T-butanol 94% 97.5% Acetone 93% 99.2%

[0050] In addition, the crude peptide (trifluoroacetate form, purity 85%) consisting of the amino acid sequence of SEQ ID NO: 4 prepared previously was subjected to the same process as the trifluoroacetate form of the crude peptide having the amino acid sequence of SEQ ID NO: 1, except that the solvent described in Table 5 was used instead of the solvent described in Table 2. As a result, the peptide (white or off-white) in trifluoroacetate form with the yield and purity described in Table 5 was obtained. The purity was confirmed by HPLC.

[0051] Solvent yield purity Methanol 88% 98.8% Ethanol 89% 98.1% Isopropanol 92% 97.6% Acetone 93% 99.3%

[0052] According to the present invention, it can be seen that crude peptides of various sizes and shapes can be purified with a purity of 95-99% without using a chromatographic method. In particular, it can be seen that peptides can be purified with a high yield (65-95%) according to the present invention, compared to the yield that usually does not even reach 50% when using a chromatographic method. At this time, it can be seen that the yield is further improved by applying a mixed solvent. In addition, it can be seen that the present invention not only overcomes the problems of the chromatographic method, but also processes a large amount of crude peptide in a non-chromatographic reactor, so that a large amount of purified peptide can be produced more efficiently.

[0053] Since the peptide purified by the purification method of the present invention naturally has a high purity, its usability is expanded to areas requiring high purity.

[0054] The present invention has the advantage of enabling the easy purification of large quantities of peptides with high yield and purity, without resorting to chromatography. Furthermore, the present invention provides peptides purified with high yield and purity. Therefore, the present invention has industrial applicability.

Claims

1. (A) A crude peptide preparation step for preparing a crude peptide in salt form; (B) a salt compound forming step of adding the above salt-formed crude peptide to a solvent and adding an acid to form a salt compound; and (C) A method for purifying a peptide in salt form, comprising a salt compound separation step of separating the salt compound from the solvent.

2. A purification method according to claim 1, wherein the salt-form crude peptide is synthesized by a solid-phase peptide synthesis method.

3. A purification method in the second paragraph, wherein the salt form is a salt form of trifluoroacetic acid.

4. A purification method in the third paragraph, wherein the acid is trifluoroacetic acid.

5. A purification method according to claim 1, wherein the solvent is a single solvent or a mixed solvent.

6. A purification method in paragraph 5, wherein the single solvent is acetone, methanol, ethanol, propanol, or butanol.

7. A purification method in paragraph 5, wherein the mixed solvent is formed by adding an auxiliary solvent to two or more of the single solvents or one or more of the single solvents.

8. A purification method in paragraph 7, wherein the auxiliary solvent is at least one selected from ethyl acetate and ethyl ether.

9. In the first paragraph, the step (B) is a purification method performed in a non-chromatographic reactor.

10. A peptide in salt form purified by any one of the purification methods of clauses 1 to 9.

Images