KR20230167201A - A preparation method of peptide and A peptide prepared using the same - Google Patents

Abstract

The present invention includes (A) crude peptide preparation step; (B) solution formation step; (C) precipitation stage; and (D) a method for producing a peptide in salt form, including the step of forming a peptide in salt form, and a peptide in salt form prepared by the production method of the present invention. The present invention has the advantage of being able to produce peptides in salt form in high yield/high purity without using chromatographic methods. In addition, the present invention has the advantage of providing salt form peptides produced in high yield/high purity.

Description

Peptide production method and peptide prepared using the same {A preparation method of peptide and A peptide prepared using the same}

The present invention relates to a method for producing peptides and peptides produced using the same. More specifically, the present invention relates to a method for easily producing peptides in salt form with high yield/high purity without using chromatography, and to peptides produced using the same. It's about.

In order to improve the stability of the peptide, it is necessary to prepare the peptide in a salt form, and if necessary, it is necessary to change the salt from one form to another.

Meanwhile, methods for chemically synthesizing peptides can be largely divided into liquid phase synthesis and solid phase synthesis. Liquid peptide synthesis is a classic synthesis method, but has the problem of difficulty in separating/purifying the target. In addition, the solid-phase peptide synthesis method developed by R. B. Merrifield in 1963 has the advantage of being automated and is widely used. However, this method also requires purification to obtain the target product of desired purity. For such purification, chromatographic methods have been conventionally used (see International Patent Application Publication No. WO 2009/098707 A1, etc.). However, when purifying peptides by chromatographic methods (e.g., reversed-phase-liquid chromatography, reversed-phase-high-performance liquid chromatography), there is a disadvantage in that the yield is not high. For example, when purifying peptides by reversed-phase-liquid chromatography or reversed-phase-high-performance liquid chromatography, the yield usually does not reach 50%, and due to the nature of the chromatographic method, the amount of purification per time is inevitably small, making it difficult for economics and productivity. there is a problem. In addition, there is no choice but to use a large amount of purified water and solvent, the freeze-drying method is mainly used to obtain the final target, which requires a lot of time and energy in addition to expensive freeze-drying equipment, and the need to maintain expensive chromatography equipment. Due to this, it has problems such as high cost, low productivity, and burden on the environment.

Therefore, there is a need to develop technology that can solve such problems.

International Patent Application Publication No. WO 2009/098707 A1, August 13, 2009, Specification

One problem that the present invention aims to solve is to provide a peptide production method that can produce peptides in salt form in high yield/high purity without using chromatography.

In addition, another problem that the present invention aims to solve is to provide a peptide in salt form that can be produced in high yield/high purity without using chromatography.

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.

The present invention includes (A) a crude peptide preparation step of preparing a crude peptide in salt form; (B) a solution forming step of dissolving the crude peptide in salt form in a solvent to form a solution; (C) a precipitation step of precipitating the peptide in free base form by adjusting the acidity of the solution formed in step (B); and (D) forming a salt peptide by adding an acid to the free base peptide to form a salt peptide.

Additionally, the present invention provides a peptide in salt form prepared by the production method of the present invention.

The present invention has the effect of being able to produce peptides in salt form in high yield/high purity without using chromatographic methods. Additionally, the present invention has the effect of providing salt form peptides produced in high yield/high purity.

1 is a flowchart for explaining an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through Examples and Preparation Examples. However, the following Examples and Preparation Examples are only for illustrating the present invention and the content of the present invention is not limited by the following Examples or Preparation Examples.

In the present invention, a peptide is a polymer in which a plurality of amino acid units are linked by a covalent bond called a peptide bond. Preferably, it refers to a polymer of a length capable of chemical synthesis. The peptide of the present invention is not limited as long as chemical synthesis is possible, but may be, for example, a peptide containing up to 50 amino acids in length.

A method for producing salt-type peptides, which is an example of the present invention, will be described with reference to FIG. 1. 1 is a flowchart for explaining an embodiment of the present invention. As shown in Figure 1, the production method as an example of the present invention includes (A) crude peptide preparation step; (B) solution formation step; (C) precipitation stage; and (D) forming a peptide in salt form.

(A) The crude peptide preparation step is to prepare the crude peptide in salt form. Crude peptides may be peptides that are subject to conversion to free base form. The crude peptide is not limited as long as it is in salt form, but may be synthesized by solid phase peptide synthesis. Peptides synthesized by solid-phase peptide synthesis may be in the salt form of trifluoroacetic acid. Accordingly, 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. In this way, by directly targeting the salt form of the crude peptide synthesized by the solid-phase peptide synthesis method, the production can be carried out more simply.

Meanwhile, the crude peptide in salt form may be a preprocessed product that has gone through a pretreatment process. Since such pretreatment can increase the purity of the peptide in salt form during the pretreatment process, the resulting free base peptide can also have a higher purity.

The pretreatment process includes (a) a pretreatment object preparation step of preparing a crude peptide in salt form, which is the pretreatment object; (b) a salt compound forming step of adding the pretreatment object 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.

In step (a), the crude peptide in salt form, which is the subject of pretreatment, is not limited as long as it is in salt form, but may be synthesized by a solid-phase peptide synthesis method. Peptides synthesized by solid-phase peptide synthesis may be in the salt form of trifluoroacetic acid. Accordingly, the salt form may be a salt form of trifluoroacetic acid. Specifically, the crude peptide in salt form, which is the subject of pretreatment, may be a crude peptide in the salt form of trifluoroacetic acid. The pretreatment object may, for example, have a purity of 90% or less.

(b) The salt compound forming step is a step of adding the pretreatment object to the solvent and adding acid to form a salt compound. At this time, 'salt compound' may refer to a peptide in salt form formed by additionally adding an acid to a crude peptide in salt form. By adding acid to the crude peptide in salt form, which is the subject of pretreatment, 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.

At this time, the solvent is not limited as long as it can dissolve the crude peptide in salt form, which is the subject of pretreatment, 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. Butanol also includes n-butanol, and/or tert-butanol. A mixed solvent may be formed by adding an auxiliary solvent to two or more single solvents or one or more single solvents. In other words, 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 co-solvent mainly promotes the salt compound to form a solid phase crystal, thereby making the separation of the salt compound easier and showing excellent 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. For example, the mixed solvent to which such an auxiliary solvent is added may be a mixture of at least one selected from acetone, methanol, ethanol, propanol, or butanol and at least one selected from ethyl acetate or ethyl ether.

The co-solvent in the mixed solvent may be preferably 1 to 50% by volume, more preferably 5 to 40% by volume, and even more preferably 10 to 40% by volume. Within this range, more effective purification is possible in terms of purity and yield.

By applying such a solvent, the peptide appears to be purified more effectively.

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 crude peptide in salt form, which is the subject of pretreatment. If it falls below that range, there is a risk that the purity will decrease, and if it exceeds the range, there is a risk that the yield will decrease.

By applying this amount of solvent, the peptide appears to be purified more effectively.

In addition, salt compound formation can be preferably carried out at -10°C to 70°C, more preferably at 5°C to 70°C, and even more preferably at 10°C to 50°C. If it falls below that range, there is a risk that the purity will decrease, and if it exceeds the range, there is a risk that the yield will decrease.

In addition, the amount of acid may be preferably 0.5 to 3 equivalents, and more preferably 1 equivalent, based on 1 equivalent of the crude peptide in salt form, which is the subject of pretreatment. Below this range, there is concern that purification may be insufficient, and if it exceeds this range, impurities may be generated and purity may be lowered.

Additionally, the crude peptide in salt form, which is the subject of pretreatment, may be in a salt form corresponding to an acid salt. For example, the crude peptide in salt form, which is the subject of pretreatment, may be a salt of trifluoroacetic acid, and the acid may be trifluoroacetic acid.

Specifically, the acid is trifluoroacetic acid, and in step (b), trifluoroacetic acid is added to the solvent to which the pretreatment object has been added, mixed at room temperature, raised to 40°C to 50°C, mixed again, and then returned to room temperature. It can be carried out including a cooling process. At this time, mixing can be performed for 0.5 hours. Additionally, remixing can be performed for 1 hour.

Additionally, step (b) may further include a final mixing process after cooling. At this time, final mixing can be performed for 1 hour.

(c) The salt compound separation step is a step of separating the salt compound from the solvent. Specifically, separation may include a process of washing and then drying the filtrate obtained by filtering the salt compound. At this time, washing can be performed with the same solvent as the solvent in step (b). The filtrate can be filtered through a filter commonly used for filtration.

The pretreatment process can be performed in a reactor other than a chromatography reactor. Specifically, step (b) may be performed in a reactor other than a chromatography reactor. At this time, the reactor may be a non-chromatographic reactor. Such a reactor may not include a stationary phase for chromatography.

(B) The solution formation step is a step of dissolving the crude peptide in salt form in a solvent to form a solution. In step (B), the solvent may be one or more selected from water, methanol, ethanol, propanol, butanol, acetone, acetonitrile, or tetrahydrofuran. Preferably, it may be one or more selected from water, methanol, ethanol, propanol, butanol, or acetonitrile. Propanol includes n-propanol and/or isopropanol. Butanol also includes n-butanol, and/or tert-butanol. At this time, the yield can be further increased by mixing two or more solvents. Preferably, it may be a mixture of water or at least one selected from methanol and acetonitrile.

Crude peptides in salt form may be acidic in solvents. That is, the crude peptide in salt form may be a peptide that is acidic (e.g., pH less than 7). For example, a peptide salt formed from an acid can act as an acid because at least the carboxyl group at the C-terminus is in a free state. Therefore, an example of a crude peptide in salt form is an acid addition salt, and the carboxyl group at the C-terminus may be a peptide in a free state.

In step (B), dissolution can be performed by adjusting the pH by adding a pH adjuster to the solvent. When the crude peptide in salt form is an acidic peptide, the preferred pH may be pH 7 to 9. The pH adjusting agent is not limited as long as it can adjust pH, and may be, for example, an alkalinizing agent. More specifically, the alkalinizing agent may be one or more selected from sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, or ammonia. Ammonia includes liquid ammonia.

The solution formed in step (B) may be a liquid filtrate obtained by dissolving the crude peptide in salt form in a solvent and filtering the dissolved product. Impurities can be removed through such filtration.

(C) The precipitation step is a step to precipitate the peptide in free base form by adjusting the acidity of the solution formed in step (B).

In step (C), acidity adjustment may be to adjust the pH of the solution to preferably pH 3.5 to 5.5. Specifically, the acidity control in step (C) may be to adjust the pH of the solution to pH 3.5 to 5.5 using a pH adjuster. The pH adjuster may be an acidifier, and acidity control can be performed by adding an acidifier as a pH adjuster to the solution. The acidifying agent may be, for example, one or more selected from acetic acid or hydrochloric acid.

In this way, it appears that by adjusting the pH in step (B) and/or step (C), acid is liberated from the salt form of the peptide, and as a result, the free base form of the peptide is precipitated.

In step (C), the precipitated free base peptide may be separated from the solution. Separation can be carried out including the process of filtering the sediment, washing the sediment filtrate, and then drying it. Washing can be done with acetic acid and water. Filtration may be by filter paper.

Step (B) and/or step (C) can be preferably carried out at -10°C to 70°C, more preferably at 5°C to 70°C, and even more preferably at 10°C to 40°C.

(D) The salt form peptide formation step is the step of adding acid to the free base peptide to form a salt form peptide.

Specifically, the formation of a peptide in salt form can be performed including the process of adding a peptide in a free base form to a solvent and adding an acid. More specifically, the formation of salt-type peptides can be performed by stirring a solvent to which acid and free-base peptides are added, then filtering and freeze-drying.

At this time, the amount of acid added may vary depending on the desired peptide, but for example, the amount of acid is preferably 0.1 to 10 equivalents, more preferably 1 to 7 equivalents, and even more per equivalent of the peptide in free base form. Preferably, it may be 1 to 4 equivalents. In this range, salt-type peptides can be formed more stably and effectively.

In step (D), the acid is a pharmaceutically acceptable organic or inorganic acid, for example, one selected from hydrochloric acid, sulfuric acid, para-toluenesulfonic acid, phosphoric acid, methanesulfonic acid, formic acid, trifluoroacetic acid, or acetic acid. You can.

In step (D), the peptide in salt form may be formed in a solvent, and the solvent may be, for example, one or more selected from water, methanol, ethanol, propanol, butanol, acetone, acetonitrile, or tetrahydrofuran. Preferably, it may be one or more selected from water, methanol, ethanol, or propanol. Propanol includes n-propanol and/or isopropanol. Butanol also includes n-butanol, and/or tert-butanol.

In step (D), the peptide in salt form can be formed at a certain pH by the added acid. For example, the salt form of the peptide can be formed at pH 1 to 7, preferably pH 1 to 6, and more preferably pH 1 to 5.

In step (D), the salt form of the peptide may be the same or different from the salt form of the crude peptide in step (A). For example, the crude peptide in the salt form of step (A) consists of a salt of trifluoroacetic acid, and the peptide in the salt form of step (D) consists of a salt of trifluoroacetic acid or a salt of non-trifluoroacetic acid. You can. As a specific example, the peptide in salt form may be composed of a salt selected from hydrochloric acid, sulfuric acid, para-toluenesulfonic acid, phosphoric acid, methanesulfonic acid, formic acid, trifluoroacetic acid, or acetic acid.

In this way, according to the present invention, the desired salt form of the peptide can be easily prepared. In particular, when the salt form of the peptide formed in step (D) is in a different salt form from the crude salt form of the peptide in step (A), it can be said to be effective in that purification and salt change can be performed simultaneously.

Each of these steps can be performed in a general reactor (e.g., a non-chromatography reactor) rather than using a chromatography device. Therefore, according to the present invention, problems associated with the application of chromatography can be avoided, and peptides in salt form can be easily produced with high efficiency and high purity.

Additionally, each step can be preferably carried out at -10°C to 70°C, more preferably at 5°C to 70°C, and even more preferably at 10°C to 50°C. If it falls below that range, there is a risk that the purity will decrease, and if it exceeds the range, there is a risk that the yield will decrease.

Additionally, the salt form of the peptide, which is an embodiment of the present invention, can be produced by the production method that is an embodiment of the present invention.

Below, through preparation examples, we will look at the preparation method and peptide, which are an example of the present invention, in more detail.

Hereinafter, the reagents and materials used in the preparation examples were commercially available and of the highest quality. Unless otherwise specified, those purchased from Sigma-aldrich were used.

<Preparation example> Preparation of peptide in salt form

1. Preparation of crude peptide prepared by solid phase peptide manufacturing method

Crude peptides in the form of trifluoroacetate listed in Table 1 were prepared by a general solid-phase peptide production method. Among them, case 1-1 is illustratively shown as follows. Specifically, it was prepared using the standard fluorenylmethyloxycarbonyl solid phase peptide synthesis (Fmoc-SPPS) method using the starting material H-Asp(OtBu)-2-ClTrt solid resin (substitution ratio: 0.63 to 0.67 mmol/g). . First, 125.0 g of the starting material, H-Asp(OtBu)-2-ClTrt peptide resin, was added to a well-dried reactor, and then N, N-dimethylformamide (DMF, 1250 ml, 10 mL/g resin) was added and incubated for 20 minutes. The resin was swollen with stirring, then filtered, the filtrate 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 compared to the equivalent of the starting material) and hydroxybenzotriazole (HOBt, 1.5 equivalents compared to the equivalent of the starting material) were added and N, N-dimethylformamide (DMF, per gram of the starting material) 4.5 mL) was added and dissolved, and then diisopropylethylamine (DIPEA, 1.5 equivalents compared to the equivalent of 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 compared to the equivalent of starting material) was added. This activated 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, the activated amino acid (Fmoc-Leu-OH) derivative solution prepared in another reactor was slowly added, and the reaction solution temperature was 10- It was stirred at 30°C for 2 hours. The ninhydrin (Kaiser) test method was used to confirm the reaction. After completion of the reaction, the peptide-resin was filtered and washed with the solvent N, N-dimethylformamide (DMF) and methyl ti-butyl ether (MTBE), respectively, and then the peptide-resin was again washed with N-dimethylformamide (DMF). The solid was washed twice (750 ml, 6 mL/g resin). The next fluorenylmethyloxycarbonyl (Fmoc) deprotection reaction is performed by adding a 5% piperidine mixed solution (v/v/w/v: 5% piperidine/1.25% DBU/1% HOBt/DMF) to the peptide-resin. ) was treated twice (750ml, 6mL/g resin, stirred for 10 minutes each and then filtered) to proceed with the deprotection reaction, and then filtered, and the filtered peptide-resin was mixed with solvent, N-dimethylformamide (DMF) x 2. Washed twice with methyl ti-butyl ether (MTBE) x 2 times and with N,N-dimethylformamide (DMF) x 2 times (60 mL/g resin for each solvent). Fluorenylmethyloxycarbonyl (Fmoc) deprotection reaction was confirmed using the ninhydrin (Kaiser) test. Coupling and fluorenylmethyloxycarbonyl (Fmoc) deprotection reactions for #3, #4, and #5 amino acid derivatives were performed in the same manner as above, that is, #3 reaction Fmoc-His(Trt)-OH, Using #4 reaction Fmoc-Leu-OH and #5 reaction Fmoc-Glu(OtBu)-OH, amino acids were sequentially reacted with peptide-resin to produce Fmoc-Glu(OtBu)1-Leu2-Hi(Trt)3- Leu4-Asp(OtBu)5-2-ClTrt peptide-resin was synthesized. However, when #3 amino acid introduces Fmoc-His(Trt)-OH, mixed solvent, N, N-dimethylformamide/dichloromethane (DCM/DMF) and N-hydroxybenzotriazole/DMF are used to activate the carboxyl group. The coupling reaction was performed using N,N,N-diisopropylcarbodiimide. The N-terminal fluorenylmethyloxycarbonyl (Fmoc)-group of the Fmoc-Glu(OtBu)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide-resin is 5% pyrrhenyl. Dean mixed solvent (v/v/w/v: 5% piperidine/1.25% DBU/1% HOBt/DMF) was added, stirred, filtered and washed to obtain H-Glu(OtBu)1-Leu2-His(Trt )3-Leu4-Asp(OtBu)5-2-ClTrt peptide-resin was synthesized.

In another reactor, add a deprotector mixed solution of trifluoroacetic acid/triisopropylsilane/dichloromethane (TFA/TIS/DCM: 55/5/40, v/v/v, 450ml, 10mL per gram of peptide resin). It was added and the temperature was cooled to 0-5°C. The obtained peptide-resin was slowly added to the cooled solution while maintaining the temperature of the reaction solution below 17°C (optimal temperature, 13.8 to 14.6°C). Subsequently, the reaction solution was heated to 23-27°C and stirred at the same temperature for 90 minutes. For the reaction solution, the solid resin was filtered using a polytetrafluoroethylene (PTFE) filter, washed twice with trifluoroacetic acid (TFA) (1.0 mL x 2 times per gram of peptide resin), and all of the filtrate was collected. The resulting filtrate was concentrated under reduced pressure at 30°C or lower until it reached 20-30% by volume. Pre-cooled methyl ti-butyl ether (MTBE) solvent (approximately 10 times the concentration volume) was added to the concentrated residue to produce the 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 ti-butyl ether (MTBE) (50% of the amount of MTBE used for precipitation) solvent. The filtered crude peptide was vacuum dried at 24°C for about 30 hours to produce a crude peptide (trifluoroacetate form, 57.4 g) having the amino acid sequence of SEQ ID NO: 1 with 98.4% deprotected peptide yield and 86% HPLC purity. synthesized. The rest other than 1-1 listed in Table 1 were synthesized using the same solid-phase peptide production method as 1-1 (see WO2011/019123 A1, US2019/0161517 A1, WO2020/184901 A1). As shown in Table 1, the synthesized crude peptide had the number of amino acids 5 to 9. Purity was confirmed by HPLC method, and amino acid sequence was confirmed by LC/MS/MS method. Additionally, the molecular weight was confirmed by LC/MS method. The results are shown in Table 1 below.

Amino acid sequence of crude peptide water(%) mass spectrometer theoretical value Analysis value 1-1
Glu-Leu-His-Leu-Asp (SEQ ID NO: 1)
86 625.7 625.4 1-2
Leu-Gln-Val-Val-Tyr-Leu-His (SEQ ID NO: 2)
89 871.0 871.1 1-3
Asp-Gly-Glu-Phe-Phe (SEQ ID NO: 3)
90 613.6 613.6 1-4
Tyr-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr (SEQ ID NO: 4)
85 785.8 785.1

2. Preparation of pretreatment material (formation and separation of salt compounds)

50 g of the previously prepared crude peptide (trifluoroacetate form, 86% purity) consisting of the amino acid sequence of SEQ ID NO: 1 was added to 500 mL of the solvent shown in Table 2 {in a non-chromatography reactor (e.g., batch reactor)}, 1 equivalent of trifluoroacetic acid (TFA) was slowly added and stirred at room temperature (25°C) for 0.5 hours. Slowly increase the temperature to 40-50℃ and stir for 1 hour. The reaction solution was cooled to room temperature (25°C) and stirred for 1 hour. Afterwards, it was filtered through filter paper, washed with 100 ml of the solvent shown in Table 2, and vacuum dried at 30°C for about 24 hours. As a result, trifluoroacetate-type peptides (white or off-white) with the yield and purity shown in Table 2 were obtained. Purity was confirmed by HPLC method.

menstruum transference number water 1-1-1 methanol 65% 99.2% 1-1-2 Methanol/ethyl ether (400/100, v/v) 75% 97.7% 1-1-3 Methanol/ethyl acetate (300/200, v/v) 74% 98.3% 1-1-4 ethanol 68% 98.9% 1-1-5 Ethanol/ethyl ether (450/50, v/v) 72% 97.8% 1-1-6 isopropanol 67% 95.2% 1-1-7 Isopropanol/ethyl ether (475/25, v/v) 75% 95.8% 1-1-8 t-butanol 73% 96.9% 1-1-9 t-butanol/ethyl ether (475/25,v/v) 79% 96.7% 1-1-10 acetone 72% 99.3%

In addition, for the previously prepared crude peptide consisting of the amino acid sequence of SEQ ID NO: 2 (trifluoroacetate form, purity 89%), the crude peptide of SEQ ID NO: 1 was used, except that the solvent listed in Table 3 was used instead of the solvent listed in Table 2. It went through the same process as trifluoroacetate of crude peptide consisting of amino acid sequence. As a result, peptides (white or off-white) in the form of trifluoroacetate were obtained with the yield and purity shown in Table 3. Purity was confirmed by HPLC method.

menstruum transference number water 1-2-1 methanol 81% 99.2% 1-2-2 Methanol/ethyl ether (400/100, v/v) 86% 98.4% 1-2-3 Methanol/ethyl acetate (300/200, v/v) 86% 98.7% 1-2-4 ethanol 83% 98.9% 1-2-5 Ethanol/ethyl ether (450/50, v/v) 85% 98.5% 1-2-6 isopropanol 88% 97.5% 1-2-7 Isopropanol/ethyl ether (475/25, v/v) 90% 97.1% 1-2-8 acetone 85% 99.4%

In addition, for the previously prepared crude peptide consisting of the amino acid sequence of SEQ ID NO: 3 (trifluoroacetate form, purity 90%), the crude peptide of SEQ ID NO: 1 was used, except that the solvent listed in Table 4 was used instead of the solvent listed in Table 2. The same process as trifluoroacetate of crude peptide with amino acid sequence was performed. As a result, peptides (white or off-white) in the form of trifluoroacetate were obtained with the yield and purity shown in Table 4. Purity was confirmed by HPLC method.

menstruum transference number water 1-3-1 methanol 90% 98.9% 1-3-2 ethanol 93% 98.5% 1-3-3 isopropanol 95% 97.8% 1-3-4 t-butanol 94% 97.5% 1-3-5 acetone 93% 99.2%

In addition, for the previously prepared crude peptide consisting of the amino acid sequence of SEQ ID NO: 4 (trifluoroacetate form, purity 85%), the crude peptide of SEQ ID NO: 1 was used, except that the solvent listed in Table 5 was used instead of the solvent listed in Table 2. The same process as trifluoroacetate of crude peptide with amino acid sequence was performed. As a result, trifluoroacetate-type peptides (white or off-white) with the yield and purity shown in Table 5 were obtained. Purity was confirmed by HPLC method.

menstruum transference number water 1-4-1 methanol 88% 98.8% 1-4-2 ethanol 89% 98.1% 1-4-3 isopropanol 92% 97.6% 1-4-4 acetone 93% 99.3%

3. Free base peptide

3-1. Production of free base peptides using crude peptides

Dissolve each of the crude peptides (1-1 to 1-4) prepared in 1. in the solvent shown in Table 6, and prepare a solution by dropping the pH adjuster shown in Table 6 at room temperature and dissolving to adjust the pH to 7 to 9. This process is carried out in a non-chromatographic reactor (e.g., batch reactor). The prepared solution is filtered through a microfilter. While stirring the filtered solution, acetic acid was slowly added dropwise at room temperature to adjust the pH to 4, and the target peptide was crystallized into a solid precipitate by stirring at the same temperature for 5 hours. The crystallized precipitate was filtered through filter paper, washed with acetic acid/water (AcOH/H ₂ O, pH 4.0) solution, and dried. As a result, peptides (white or off-white) in free base form with the yield and purity shown in Table 6 were obtained. Purity was confirmed by HPLC method. When checking purity by HPLC method, it was also confirmed whether it was in free base form.

menstruum pH regulator transference number water 1-1A water sodium hydroxide 62% 98.1% 1-1B Water/acetonitrile (80/20, v/v) ammonia 68% 98.3% 1-2A water sodium hydroxide 65% 98.6% 1-2B Water/acetonitrile (80/20, v/v) ammonia 67% 99.1% 1-3A water sodium hydroxide 72% 98.4% 1-3B Water/acetonitrile (80/20, v/v) ammonia 75% 98.2% 1-4A water sodium hydroxide 76% 97.8% 1-4B Water/acetonitrile (80/20, v/v) ammonia 78% 98.0%

3-2. Production of free base peptides using pretreated products

Dissolve each of the pretreatment materials (1-1-2, 1-2-6, 1-3-3, 1-4-3) prepared in 2. in the solvent shown in Table 7, and add the pH adjuster shown in Table 7 at room temperature. Add dropwise and dissolve to adjust pH to 7~9 to prepare a solution. This process is carried out in a non-chromatographic reactor (e.g., batch reactor). The prepared solution is filtered through a microfilter. While stirring the filtered solution, acetic acid was slowly added dropwise at room temperature to adjust the pH to 4, and the target peptide was crystallized into a solid precipitate by stirring at the same temperature for 5 hours. The crystallized precipitate was filtered through filter paper, washed with acetic acid/water (AcOH/H ₂ O, pH 4.0) solution, and dried. As a result, peptides (white or off-white) in free base form with the yield and purity shown in Table 7 were obtained. Purity was confirmed by HPLC method. When checking purity by HPLC method, it was also confirmed whether it was in free base form.

menstruum pH regulator transference number water 1-1-2A water sodium hydroxide 74% 99.2% 1-1-2B Water/acetonitrile (80/20, v/v) ammonia 76% 99.5% 1-2-6A water sodium hydroxide 78% 99.1% 1-2-6B Water/acetonitrile (80/20, v/v) ammonia 79% 99.3% 1-3-3A water sodium hydroxide 76% 98.8% 1-3-3B Water/acetonitrile (80/20, v/v) ammonia 79% 99.0% 1-4-3A water sodium hydroxide 80% 99.1% 1-4-3B Water/acetonitrile (80/20, v/v) ammonia 83% 99.2%

4. Preparation of salt-type peptide

The free base peptides (1-1-2A, 1-2-6A, 1-3-3A, 1-4-3A) prepared in 3 were added to 10 times the amount of solvent (purified water), and the results are shown in Table 8. The acid described (1 to 3 equivalents per equivalent of peptide) was added slowly and stirred at room temperature for 3 hours. After the stirring was completed, insoluble substances were removed by filtering through filter paper, and the filtered liquid was freeze-dried. As a result, salt form peptides (white or off-white) were obtained with a yield of over 95% and a purity of over 99%. This is because the yield is not lowered due to the nature of the freeze-drying method, and the purity of the freeze-dried object (peptide in free base form) is maintained even in the freeze-dried product. In other words, since free base peptides can be obtained with high purity, salt form peptides can also be obtained with high purity.

mountain acid equivalent 1-1-2A1 Hydrochloric acid 2 1-1-2A2 sulfuric acid One 1-1-2A3 p-toluenesulfonic acid 2 1-1-2A4 Methanesulfonic acid 2 1-1-2A5 phosphoric acid 2 1-1-2A6 formic acid 3 1-1-2A7 Trifluoroacetic acid 2 1-1-2A8 acetic acid 3 1-2-6A1 Hydrochloric acid 2 1-2-6A2 sulfuric acid One 1-2-6A3 p-toluenesulfonic acid 2 1-2-6A4 Methanesulfonic acid 2 1-2-6A5 phosphoric acid 2 1-2-6A6 formic acid 3 1-2-6A7 Trifluoroacetic acid 2 1-2-6A8 acetic acid 3 1-3-3A1 Hydrochloric acid 2 1-3-3A2 sulfuric acid One 1-3-3A3 p-toluenesulfonic acid 2 1-3-3A4 Methanesulfonic acid 2 1-3-3A5 phosphoric acid 2 1-3-3A6 formic acid 3 1-3-3A7 Trifluoroacetic acid 2 1-3-3A8 acetic acid 3 1-4-3A1 Hydrochloric acid 2 1-4-3A2 sulfuric acid One 1-4-3A3 p-toluenesulfonic acid 2 1-4-3A4 Methanesulfonic acid 2 1-4-3A5 phosphoric acid 2 1-4-3A6 formic acid 3 1-4-3A7 Trifluoroacetic acid 2 1-4-3A8 acetic acid 3

From the above results, it can be seen that crude peptides (including preprocessed products) of various sizes and shapes can be obtained with high purity (approximately 99% or more) and various types of salt-type peptides without using chromatographic methods. In addition, since free base peptides with improved yield can be obtained by applying a mixed solvent, various types of target salt peptides can also be produced in higher yields using the same. In particular, compared to the yield usually less than 50% when using chromatographic methods, peptides in free base form can be obtained in high yield according to the present invention, and as a result, various types of desired salt form peptides can be manufactured. You can see that it can be done. In addition, according to the present invention, not only can the problems of the chromatography method be overcome, but also a large amount of crude peptides (including preprocessed products) can be processed in a non-chromatographic reactor, so a large amount of salt-type peptides can be produced more easily. It can be seen that it can be manufactured efficiently.

Since the salt form of the peptide prepared by the method of the present invention naturally has high purity, its utility is expanded to areas where high purity is required.

<110> ENSOL BIOSCIENCES, INC. <120> A preparation method of peptide and A peptide prepared using the same <130>GP22028 <160> 4 <170>CopatentIn 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 1 Glu Leu His Leu Asp 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 2 Leu Gln Val Val Tyr Leu His 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 3 Asp Gly Glu Phe Phe 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 4 Tyr Gly Ala Gly Ala Gly Ala Gly Tyr 1 5

Claims (12)

(A) Crude peptide preparation step of preparing crude peptide in salt form;
(B) a solution forming step of dissolving the crude peptide in salt form in a solvent to form a solution;
(C) a precipitation step of precipitating the peptide in free base form by adjusting the acidity of the solution formed in step (B); and
(D) A method for producing a peptide in salt form, comprising the step of forming a peptide in salt form by adding an acid to the peptide in free base form to form a peptide in salt form. The method of claim 1, wherein the crude peptide in salt form is synthesized by a solid-phase peptide synthesis method. The method of claim 1, wherein the peptide in salt form consists of a salt selected from hydrochloric acid, sulfuric acid, para-toluenesulfonic acid, phosphoric acid, methanesulfonic acid, formic acid, trifluoroacetic acid, or acetic acid. The method of claim 1, wherein the solvent is one or more selected from water, methanol, ethanol, propanol, butanol, acetone, acetonitrile, or tetrahydrofuran. The method of claim 1, wherein in step (B), the dissolution is performed by adjusting the pH by adding a pH adjuster to the solvent. The method of claim 5, wherein the pH adjuster is an alkalinizing agent. The method of claim 1, wherein in step (C), the pH of the solution is adjusted to pH 3.5 to 5.5 using a pH adjuster. The method of claim 7, wherein the pH adjuster is an acidifying agent. The method of claim 1, wherein the production method is performed in a non-chromatographic reactor. The method of claim 1, wherein the crude peptide in salt form is a pre-processed product that has undergone a pretreatment process. According to clause 10,
The preprocessing process is
(a) a pretreatment object preparation step of preparing a crude peptide in salt form as a pretreatment object;
(b) a salt compound forming step of adding the pretreatment object to a solvent and adding an acid to form a salt compound; and
(c) A method for producing a peptide in salt form, including a salt compound separation step of separating the salt compound from the solvent. A peptide in salt form prepared by the production method of any one of claims 1 to 11.