WO2025089467A1 - Method for producing salt-type modified peptide and peptide produced using same - Google Patents

Abstract

The present invention provides a method for producing a salt-type modified peptide and a salt-type modified peptide, wherein the method comprises (A) a salt-type peptide preparation step of preparing a peptide in a salt form; and (b) a step of contacting the peptide with a solvent to form a salt-type modified peptide. The method of the present invention has an advantage in that peptides in a high purity state can be easily prepared. In addition, the peptide of the present invention can be advantageously prepared in a high purity state.

Description

Method for producing a salt-form modified peptide and peptide produced using the same

The present invention relates to a method for producing a peptide whose salt form is modified and a peptide produced using the same, and more specifically, to a method for facilitating the modification of a peptide's salt form and producing a peptide in a high purity state, and a peptide that can be produced in a high purity state.

Peptides are mainly synthesized using the solid phase peptide synthesis (SPPS) method developed by R. B. Merrifield in 1963. The crude peptide salt synthesized by this method is manufactured in the form of trifluoroacetic acid salt (see International Patent Application Publication No. WO 2012/002668 A2, WO 2011/019123 A1). In this case, depending on the peptide, the acid equivalent exceeds 1 per 1 equivalent of free peptide.

The inventors of the present invention invented an improvement technique while studying such peptide salts.

[Prior art literature]

[Patent Document]

(Patent Document 1) International Patent Application Publication No. WO 2012/002668 A2, January 5, 2012, Specification

(Patent Document 2) International Patent Application Publication No. WO 2011/019123 A1, February 17, 2011, Specification

One problem that the present invention seeks to solve is to provide a method for producing a salt-form modified peptide capable of increasing the purity of the obtained product.

In addition, another problem that the present invention seeks to solve is to provide a peptide capable of increasing purity.

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 provides a method for producing a salt-form modified peptide, comprising: (A) a salt-form peptide preparation step of preparing a peptide in salt form; and (B) a step of contacting the peptide with a solvent to form a salt-form modified peptide, wherein in the step (A), the peptide includes a basic amino acid, and the basic amino acid is histidine, and in the step (B), the solvent is at least one selected from alcohol and acetone.

In addition, the present invention provides a peptide manufactured by the manufacturing method of the present invention.

The method of the present invention has the effect of easily producing a peptide in a highly pure state. In addition, the peptide of the present invention has the effect of being able to be produced in a highly pure state.

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

Hereinafter, the present invention will be described in more detail by examples and manufacturing examples. However, the following examples and manufacturing examples are only intended to illustrate the present invention, and the content of the present invention is not limited by the following examples or manufacturing 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 having a length that allows chemical synthesis. The peptide of the present invention may be a peptide having a length that includes, for example, up to 50 amino acids, as long as chemical synthesis is possible, but is not limited thereto.

In addition, in the present invention, 'salt form modification' means that the equivalent of an acid forming a salt is changed with respect to 1 equivalent of a free peptide. At this time, the change in the equivalent of the acid does not exclude the case where the equivalent of the acid forming a salt with respect to 1 equivalent of a free peptide is 0. In other words, a transformation from a salt form to a non-salt form may also be included in the salt form modification.

A method for producing a salt-form modified peptide, which is one embodiment of the present invention, is described with reference to FIG. 1. FIG. 1 is a flow chart for explaining one embodiment of the present invention. As shown in FIG. 1, the method for producing a salt-form modified peptide, which is one embodiment of the present invention, includes (A) a salt-form peptide preparation step; and (B) a salt-form modified peptide formation step.

Step (A) is a step for preparing a salt-form peptide as a target of salt-form modification. At this time, the target peptide includes a basic amino acid, and the basic amino acid is histidine. In this way, by contacting a specific peptide including histidine among basic amino acids with a specific solvent, the equivalent of an acid forming a salt can be changed, and a salt-form modified peptide formed with a changed equivalent of the acid can be manufactured with higher purity. This is thought to be because the production of impurities is reduced due to a change in the equivalent of the acid, more preferably a decrease. Accordingly, the salt-form peptide of step (A) may have an additional added acid equivalent of at least 1 compared to the salt-form modified peptide of step (B). That is, it seems that the production of impurities can be reduced and the purity can be increased by forming a salt-form modified peptide in which the equivalent of the added acid is reduced compared to the salt-form peptide. That is, it can be said that one embodiment of the present invention is to increase the purity of a obtained product by modifying a salt form.

Preferably, the salt form peptide may include at least one selected from glutamine (Gln, Q) or glutamic acid (Glu, E) located at the N-terminus. At this time, the glutamine is not limited to a position included in the peptide. In particular, in such a salt form peptide, by forming a salt form modified peptide in which the equivalent amount of the added acid is reduced, a high-purity product can be obtained more effectively.

The acid may be, but is not limited to, for example, trifluoroacetic acid, hydrochloric acid, p-toluenesulfonic acid, methanesulfonic acid, sulfuric acid, phosphoric acid, or formic acid.

The salt form modified peptide can be in a mono-salt form or a non-salt form, i.e., a form in which 1 to 0 equivalents of acid are added, as a result of reducing the equivalents of the acid added.

The salt-form modified peptide may be, for example, a trifluoroacetic acid monosalt (TFA monosalt) of a peptide consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. At this time, the monosalt indicates that TFA forms a salt in a ratio of 0.7 to 1.3 equivalents, preferably 0.9 to 1.1 equivalents, and more preferably 1 equivalent, per 1 equivalent of free peptide. Such a salt-form modified peptide can be manufactured with high purity with a low impurity content. Therefore, it exhibits properties more suitable for a finished pharmaceutical product.

On the other hand, the salt-form peptide can exhibit acidity. That is, the salt-form peptide can be a peptide exhibiting acidity (e.g., pH less than 7). For example, a peptide salt formed from an acid can act as an acid in a solvent because at least the carboxyl group at the C-terminus is in a free state. Therefore, an example of a salt-form peptide can be a peptide that is an acid addition salt and has a free carboxyl group at the C-terminus.

Specifically, the peptide of step (A) 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 salt-form peptide may be a peptide that is a salt form of trifluoroacetic acid. In this case, trifluoroacetic acid may be 2 equivalents per 1 equivalent of free peptide. By directly processing the peptide in the salt form synthesized by the solid-phase peptide synthesis method in this way, manufacturing can be carried out more simply.

Meanwhile, the salt-form peptide may be a preprocessed product that has undergone a preprocessing process. Such a preprocessed product can further increase the purity of the salt-form peptide during the preprocessing process, and thus the resulting salt-form modified peptide can also have a higher purity.

The pretreatment process can be performed using a traditional chromatographic purification method.

(B) Step is a step of forming a salt-form modified peptide by contacting a peptide in salt form with a specific solvent. The specific solvent is at least one selected from alcohol or acetone. At this time, the alcohol may be at least one selected from methanol, ethanol, or propanol, for example. In addition, the propanol may be at least one selected from n-propanol or isopropanol, for example.

In step (B), contact may be by mixing, etc., and may preferably be performed at 20°C to 50°C.

In addition, in step (B), the solvent may be 500 to 1500 parts by weight per 100 parts by weight of the peptide in salt form. 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.

In step (B), the salt-form modified peptide can be formed through additional processes of filtration, washing, and drying after contact.

In addition, a peptide according to an embodiment of the present invention can be manufactured by a manufacturing method according to an embodiment of the present invention.

For example, a peptide according to one embodiment of the present invention may be a trifluoroacetic acid monosalt of a peptide consisting of an amino acid sequence of SEQ ID NO: 1 (ELHLD) or an amino acid sequence of SEQ ID NO: 2 (LQVVYLH).

In the amino acid sequence, E represents glutamic acid (Glu), L represents leucine (Leu), H represents histidine (His), D represents aspartic acid (Asp), Q represents glutamine (Gln), V represents valine (Val), and Y represents tyrosine (Tyrosine).

The amino acids that constitute such peptides include L-forms, D-forms, and DL-forms, and the amino acids that constitute the peptide of the present invention include all of these.

At this time, the single salt indicates that trifluoroacetic acid (TFA) forms a salt in a ratio of 0.7 to 1.3 equivalents, preferably 0.9 to 1.1 equivalents, and more preferably 1 equivalent, per 1 equivalent of the free peptide. Such a peptide may have a low impurity content. Therefore, it exhibits properties more suitable for a finished pharmaceutical product.

Unless otherwise stated, the matters mentioned in the manufacturing method and peptide of the present invention are applied equally to each other to the extent of identity, unless they are contradictory.

The manufacturing method and peptide, which are embodiments of the present invention, will be examined in more detail through the following manufacturing examples and reference examples.

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

<Manufacturing Example 1> Manufacturing of salt-form modified peptide I

1.1. Preparation of salt-form peptides manufactured by the solid-phase peptide manufacturing method

Trifluoroacetic acid salts (Glu-Leu-His-Leu-Asp 2TFA, Leu-Gln-Val-Val-Tyr-Leu-His 2TFA) of peptides consisting of the amino acid sequences described in Table 1 (containing histidine as a basic amino acid) 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 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 placed in a well-dried reactor, and N,N-dimethylformamide (DMF, 1250 mL, 10 mL/g resin) was added. The resin was swelled while stirring for 20 minutes, then filtered, and 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 relative to the starting material) and hydroxybenzotriazole (HOBt, 1.5 equivalents relative to the starting material) were charged and dissolved in N,N-dimethylformamide (DMF, 4.5 mL per gram of starting material), followed by addition of diisopropylethylamine (DIPEA, 1.5 equivalents relative to the starting material). The reaction solution was cooled to 0-5°C, and 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazole-1-oxide hexafluorophosphate (HBTU, 1.5 equivalents relative to 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 the activated amino acid (Fmoc-Leu-OH) derivative solution prepared in another reactor was slowly added while stirring, 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 completion of the reaction, the peptide-resin was filtered and washed with the solvents N, N-dimethylformamide (DMF) and methyl tert-butyl ether (MTBE), 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, stirred for 10 min each and then filtered), followed by filtration. The filtered peptide-resin was washed with the solvent N,N-dimethylformamide (DMF) x 2 times, methyl tert-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 using 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 described above, that is, #3 reaction Fmoc-His(Trt)-OH, #4 reaction Fmoc-Leu-OH, and #5 reaction Fmoc-Glu(OtBu)-OH were used to sequentially react amino acids with the peptide-resin to synthesize 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 Fmoc-Glu(OtBu)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide-resin was synthesized by adding 5% piperidine mixed solvent (v/v/w/v: 5% piperidine/1.25% DBU/1% HOBt/DMF), stirring, filtering, and washing to H-Glu(OtBu)1-Leu2-His(Trt)3-Leu4-Asp(OtBu)5-2-ClTrt peptide-resin.

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 the temperature was 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). Then, 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, which was washed twice with trifluoroacetic acid (TFA) (1.0 mL per gram of peptide resin x 2 times), and the filtrates were combined. The combined filtrates were concentrated under reduced pressure at below 30°C until the volume became 20-30%. To the concentrated residue, pre-cooled methyl tert-butyl ether (MTBE) solvent (approximately 10 times the concentrated volume) was added, to generate 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 tert-butyl ether (MTBE) solvent (50% of the amount of MTBE used for precipitation). The filtered crude peptide was vacuum-dried at 24°C for about 30 hours to synthesize a crude peptide (trifluoroacetate form, 57.4 g) having an 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 for 1-1 described in Table 1 were also synthesized using a conventional solid-phase peptide production method in the same manner as 1-1 (see WO2011/019123 A1). The purity and trifluoroacetic acid equivalent were confirmed by the HPLC method, and the amino acid sequence was confirmed by the LC/MS/MS method. In addition, the molecular weight was confirmed by the LC/MS method. The results are shown in Table 1 below.

Amino acid sequence of peptide water(%) TFA
equivalent weight TFA
HPLC content (%) Theoretical value Analysis value 1-1
Glu-Leu-His-Leu-Asp(SEQ ID NO: 1)
86 2 26.7 25.1 1-2
Leu-Gln-Val-Val-Tyr-Leu-His(SEQ ID NO: 2)
89 2 20.8 21.0

1.2. Formation of salt-type modified peptides

5 g of the previously prepared 1-1 peptide (Glu-Leu-His-Leu-Asp 2 TFA, purity 86%) was added to 50 mL of the solvent described in Table 2 {in a non-chromatographic reactor (e.g., batch reactor)} and stirred at room temperature (25°C) for 0.5 hour. The temperature was slowly increased to 40 to 50°C and stirred for 1 hour. The reaction solution was cooled to room temperature (25°C) and stirred for 1 hour. Thereafter, it was filtered through a filter paper, washed with 10 mL of the solvent described in Table 2, and vacuum-dried at 30°C for about 24 hours. As a result, a white or off-white peptide (Glu-Leu-His-Leu-Asp 1TFA) having the yield and purity described in Table 2 was obtained. The purity and trifluoroacetic acid content were confirmed by the HPLC method.

menstruum transference number% water% TFA HPLC content %
TFA equivalent Theoretical value Analysis value 1-1-1 Methanol 71 98.1 13.4 12.8 1 1-1-2 Methanol/acetone (30/20, v/v) 74 97.8 13.4 13.1 1 1-1-3 Ethanol 73 97.5 13.4 13.5 1 1-1-4 Ethanol/acetone (30/20, v/v) 74 98.1 13.4 13.4 1 1-1-5 Isopropanol 78 97.2 13.4 13.2 1 1-1-6 Isopropanol/acetone (30/20, v/v) 75 97.5 13.4 13.5 1 1-1-7 N-Propanol 77 97.3 13.4 13.2 1 1-1-8 N-propanol/acetone (30/20, v/v) 79 97.7 13.4 13.4 1 1-1-9 Acetone 78 98.4 13.4 13.7 1

Also, for the previously prepared 1-2 peptide (Leu-Gln-Val-Val-Tyr-Leu-His·2TFA, purity 89%), the same process as for the 1-1 peptide was performed, except that the solvent described in Table 3 was used instead of the solvent described in Table 2. As a result, a white or off-white peptide (Leu-Gln-Val-Val-Tyr-Leu-His·1TFA) having the yield and purity described in Table 3 was obtained. The purity and the content of trifluoroacetic acid were confirmed by the HPLC method.

menstruum transference number% water% TFA HPLC content %
TFA equivalent Theoretical value Analysis value 1-2-1 Methanol 82 98.3 10.4 10.3 1 1-2-2 Methanol/acetone (30/20, v/v) 84 98.2 10.4 10.4 1 1-2-3 Ethanol 84 98.5 10.4 10.2 1 1-2-4 Ethanol/acetone (30/20, v/v) 85 97.1 10.4 10.4 1 1-2-5 Isopropanol 81 97.8 10.4 10.3 1 1-2-6 Isopropanol/acetone (30/20, v/v) 88 97.6 10.4 10.5 1 1-2-7 N-Propanol 84 97.7 10.4 10.5 1 1-2-8 N-propanol/acetone (30/20, v/v) 86 97.9 10.4 10.4 1 1-2-9 Acetone 85 98.7 10.4 10.6 1

From the above results, it can be seen that, according to the present invention, a high-purity synthetic product is obtained by forming a salt-form modified peptide in which the acid equivalent is changed compared to before modification by applying a specific solvent to a specific peptide. In addition, it can be seen that a salt-form modified peptide in which the acid equivalent is changed compared to before modification corresponds to a peptide that can be manufactured in a high-purity state.

<Manufacturing Example 2> Manufacturing of salt-form modified peptide II

2.1. Preparation of salt form peptide (Glu-Leu-His-Leu-Asp·2 hydrochloride)

10.0 g of the previously prepared 1-1 peptide (Glu-Leu-His-Leu-Asp 2 TFA, purity 86%) was dissolved in 50 mL of purified water {in a non-chromatographic reactor (e.g., batch reactor)}, and 1 N sodium hydroxide solution was added dropwise at room temperature (25°C) to adjust the pH to 7-9 to prepare a solution. The prepared solution was 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 solution was stirred at the same temperature for 5 hours to crystallize the target peptide as a solid precipitate. The crystallized precipitate was filtered through a filter paper, washed with an acetic acid/water (AcOH/H ₂ O, pH 4.0) solution, and dried to obtain the peptide (7.2 g) in the form of a free base. 7.2 g of the dried free base form of the peptide was added to 72 mL of purified water, and 3 equivalents of concentrated hydrochloric acid were slowly added at room temperature, and stirred for 30 minutes. The reaction solution was freeze-dried to obtain a white peptide (Glu-Leu-His-Leu-Asp dihydrochloride). The purity was confirmed by HPLC, and the equivalent amount of hydrochloric acid was confirmed by acid-base titration. The results are shown in Table 4.

Amino acid sequence of peptide water(%) Hydrochloric acid equivalent 2-1
Glu-Leu-His-Leu-Asp(SEQ ID NO: 1)
95.1% 2

2.2. Formation of salt-type modified peptides

5 g of the previously prepared 2-1 peptide (Glu-Leu-His-Leu-Asp 2 hydrochloride, purity 95.1%) was added to 50 mL of the solvent described in Table 5 {in a non-chromatographic reactor (e.g., batch reactor)} and stirred at room temperature (25°C) for 0.5 hour. The temperature was slowly increased to 40 to 50°C and stirred for 1 hour. The reaction solution was cooled to room temperature (25°C) and stirred for 1 hour. Thereafter, the solution was filtered through a filter paper, washed with 20 mL of the solvent described in Table 5, and vacuum-dried at 30°C for about 24 hours. As a result, a white or off-white peptide (Glu-Leu-His-Leu-Asp 1 hydrochloride) having the yield and purity described in Table 5 was obtained. Purity was confirmed by HPLC method, and the hydrochloric acid content was confirmed by acid-base titration method.

menstruum transference number% water% Hydrochloric acid equivalent 2-1-1 Acetone 89 99.2 1

From the above results, it can be seen that, regardless of the acid forming the salt, the present invention forms a salt-form modified peptide in which the equivalent amount of the acid is changed compared to before modification, thereby obtaining a high-purity synthetic product.

<Reference Example 1> Manufacturing of peptides that maintain salt form

5 g of the previously prepared 1-1 peptide (Glu-Leu-His-Leu-Asp 2TFA, purity 86%) was added to 50 mL of the solvent described in Table 6 {in a non-chromatographic reactor (e.g., batch reactor)} and stirred at room temperature (25°C) for 0.5 hour. The temperature was slowly increased to 40 to 50°C and stirred for 1 hour. The reaction solution was cooled to room temperature (25°C) and stirred for 1 hour. Thereafter, when a solvent other than water was used, the solution was filtered through a filter paper, washed with 20 mL of the solvent described in Table 6, and vacuum-dried at 30°C for about 24 hours. When water was used as the solvent, freeze-drying was performed. As a result, a white or off-white peptide (Glu-Leu-His-Leu-Asp 2TFA) having the yield and purity described in Table 6 was obtained. Purity and trifluoroacetic acid content were confirmed by HPLC method.

menstruum transference number% water% TFA equivalent 1-1-A Acetonitrile 98 88 2 1-1-B Ethyl acetate 99 87 2 1-1-C water 98 86 2

Also, for the previously prepared 1-2 peptide (Leu-Gln-Val-Val-Tyr-Leu-His·2 TFA, purity 89%), the same process as for the 1-1 peptide was performed, except that the solvent described in Table 7 was used instead of the solvent described in Table 6. As a result, a white or off-white peptide (Leu-Gln-Val-Val-Tyr-Leu-His·2TFA) having the yield and purity described in Table 7 was obtained. The purity and trifluoroacetic acid content were confirmed by the HPLC method.

menstruum transference number% water% TFA equivalent 1-2-A Acetonitrile 97 89 2 1-2-B Ethyl acetate 98 90 2 1-2-C water 97 89 2

From the above results, it can be seen that when a solvent other than the solvent applied to the present invention is applied, a salt-form modified peptide cannot be formed and a high-purity synthetic product cannot be obtained.

Finally, according to the present invention, it can be seen that a high-purity synthetic product is obtained by forming a salt-form modified peptide in which the acid equivalent weight is changed compared to before modification by applying a specific solvent to a specific peptide. The salt-form modified peptide obtained in this way (e.g., a trifluoroacetic acid monosalt of a peptide consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2) has high purity, and thus its utility is expanded to fields requiring high purity (e.g., pharmaceutical manufacturing field).

The method of the present invention has the effect of easily producing a peptide in a highly pure state. In addition, the peptide of the present invention has the effect of being able to be produced in a highly pure state. Therefore, the present invention has industrial applicability.

Claims (6)

(A) a salt form peptide preparation step for preparing a peptide in salt form; and (B) a step of contacting the peptide with a solvent to form a salt form modified peptide, In the above step (A), the peptide comprises a basic amino acid, and the basic amino acid is histidine. A method for producing a salt-form modified peptide, wherein in the step (B) above, the solvent is at least one selected from alcohol and acetone. In the first paragraph, a method for producing a salt-form modified peptide, wherein the peptide in the salt form of step (A) has an additional added acid equivalent of at least 1 compared to the salt-form modified peptide in step (B). In the first paragraph, the salt form modified peptide of the step (B) is a method for producing a salt form modified peptide in a mono-salt form or a non-salt form. A method for producing a salt-form modified peptide in claim 1, wherein the alcohol is at least one selected from methanol, ethanol, and propanol. A peptide manufactured by the manufacturing method of any one of claims 1 to 4. In claim 5, the peptide is a trifluoroacetic acid monosalt of a peptide consisting of an amino acid sequence of sequence number 1 or sequence number 2.

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