Purification method of teduglutide

Abstract

The invention relates to the field of preparation of teduglutide, in particular to a purification method of teduglutide. The method comprises the following specific steps: dissolving a teduglutide crude product in a buffer solvent, stirring at 35-45 DEG C after dissolving, and filtering to obtain a teduglutide crude product solution; performing HPLC (High Performance Liquid Chromatography) purification on the crude product solution, and collecting fractions of which the purity is greater than And adding the same volume of purified water into the fraction, and carrying out salt conversion, concentration and freeze-drying to obtain the refined teduglutide. According to the purification method of teduglutide provided by the invention, the dissolving capacity of the crude peptide in the buffer solvent is greatly improved, and the concentration of the crude product can reach about 40mg / mL; the purification mode is simple, a buffer solvent is directly used as a mobile phase A, a product with the purity of 99% can be obtained through one-step purification, and the purification period is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the field of the preparation of teduglutide, and specifically relates to a purification method of teduglutide. Background Art

[0002] Teduglutide is a glucagon-like peptide-2 (GLP-2) analogue, mainly used for the treatment of short bowel syndrome (SBS). This drug has performed excellently in clinical trials, with remarkable efficacy and high safety, greatly improving the quality of life of patients. With the in-depth research on the treatment of short bowel syndrome with teduglutide and the expansion of clinical applications, more patients will benefit from this treatment method, and teduglutide will be further optimized and used in the future. The purity of teduglutide plays a crucial role in its quality and effect.

[0003] Currently, there are many synthetic methods for teduglutide, but there are relatively few methods for optimizing the purity of teduglutide. This is mainly because teduglutide is not easily soluble in solution, often precipitates repeatedly, and is not easily soluble after precipitation. Although the existing purification methods of teduglutide can improve the purity, the effect is still not ideal. For example, in the Chinese patent with the publication number CN104045707A, an acetic acid acetonitrile aqueous solution is selected for dissolution, and after dissolution, purified water is added to dilute to 10 - 30% of the total volume of acetic acid and acetonitrile. Purification is carried out in two steps. The first pure mobile phase is a mixture of sulfuric acid and perchloric acid. After purification, it needs to be crystallized and then redissolved, followed by ammonium acetate salt conversion, acetic acid elution, concentration, and freeze-drying. This method has low solubility of the crude product, a large sample loading volume, and the perchloric acid used in the first pure mobile phase is an explosive precursor reagent, and the sample will precipitate after standing overnight, which is not conducive to industrial production. After the first purification, crystallization treatment is required, and the operation is cumbersome. In the case in the patent with a crude product purity similar to that used in this experiment, the purification yield is 87%, which is quite different from the reproduced data of 58.3%. Another example is the Chinese patent with the publication number CN112175067A, which selects a stationary phase of 20mm * 250mm conventional C18 and a mobile phase of 0.1 - 0.3% triethylamine solution adjusted to pH 2.5 with phosphoric acid for the first step of reverse purification. It is found that the sample at the peak top precipitates severely and cannot be redissolved. In the Chinese patent with the publication number CN116003574A, 315 mg of teduglutide acetate fine peptide with a purity of 99.34% is obtained after purification, and the purification yield is 52.5%. Although this condition can obtain a teduglutide finished product with a purity of more than 99%, the concentration of the crude product is low, the purification steps are numerous, and the mobile phase is relatively complex. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides a purification method of teduglutide, which greatly improves the dissolution ability of the crude peptide of teduglutide, and the sample is stable and does not precipitate after standing for several days; the purification and salt conversion steps are simple, without other complex processes, reducing the purification cost and increasing the yield.

[0005] To achieve the object of the present invention, the present invention provides the following technical solutions: A method for purifying teduglutide, comprising the following steps: (1) Dissolve the crude teduglutide in a buffer solvent, stir at 35-45 °C after dissolution, and then filter to obtain a crude teduglutide solution; (2) Purify the crude teduglutide solution by HPLC, and collect the fractions with a purity greater than 98%; (3) Add an equal volume of purified water to the fraction, perform salt conversion, concentration, and freeze-drying to obtain the purified teduglutide; Wherein, the buffer solvent is composed of X and Y mixed in a ratio of 90-95:5-10; X is selected from one or more of DMSO, DMF, HAC, phosphoric acid, ammonia water, triethylamine, sodium hydroxide, H2O, diammonium hydrogen phosphate, Tris, citric acid, and ammonium bicarbonate; Y is selected from one or more of acetonitrile, methanol, isopropanol, and tetrahydrofuran.

[0006] According to a preferred embodiment of the present invention, in the step (1), the stirring time is 3-5 hours; more preferably, the stirring time is 4 hours.

[0007] According to a preferred embodiment of the present invention, in the step (1), the stirring temperature is 40 °C.

[0008] According to a preferred embodiment of the present invention, in the step (1), the volume ratio of the buffer solvents X and Y is 95:5.

[0009] According to a preferred embodiment of the present invention, in the mobile phase, in the step (2), the chromatographic conditions for HPLC purification are: Chromatographic column: C8 column, column length: 100-250 mm; Mobile phase A: buffer solvent; Mobile phase B: a mixture of mobile phase A and pure acetonitrile with a volume ratio of 30:70; Time and mobile phase gradient: The preparation gradient B% is 30%-38%, and the preparation time is 60 min; Flow rate: 250 mL / min; Sample injection volume: 15 g.

[0010] According to another preferred embodiment of the present invention, the column length of the chromatographic column during HPLC purification is 150 nm.

[0011] According to a preferred embodiment of the present invention, the detector used during HPLC purification is an ultraviolet detector, and the detection wavelength is 220 nm.

[0012] According to a preferred embodiment of the present invention, the chromatographic conditions for salt transfer in step (3) are as follows: Chromatographic column: C8 column, column length: 100 - 250 mm; Mobile phase: A 0.3% aqueous acetic acid solution, B acetonitrile, C 50 mM ammonium acetate; Time and mobile phase gradient: Salt transfer: B% is 5%, C% is 95%; preparation time is 14 - 23 min; Elution: B% is 5%, A% is 95%; preparation time is 14 - 23 min; B% is 70%, A% is 30%, gradient time is 50 min; Flow rate: 250 mL / min; Sample injection volume: 32.5 g.

[0013] According to another preferred embodiment of the present invention, the preparation time for salt transfer and elution is 14 min.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The purification method of tirzepatide provided by the present invention greatly improves the solubility of the crude peptide in the buffer solvent, and the concentration of the crude product can reach about 40 mg / mL; moreover, the purification method is simple. The buffer solvent is directly used as mobile phase A, and a product with a purity of 99% can be obtained through one-step purification, greatly shortening the purification cycle.

[0015] 2. The purification method of tirzepatide provided by the present invention has simple purification and salt transfer steps, without other complex processes, facilitating later large-scale production; it has the effects of improving the purification yield, reducing the purification cost, and increasing the yield.

[0016] 3. In the prior art, tirzepatide is not easily soluble in solution, and there are often phenomena of repeated precipitation and difficulty in redissolving after precipitation. The main reasons are that in a conventional solution, the hydrophilic groups are not easily unfolded, the hydrophobic groups between molecules attract each other to cause polymerization, and the spatial form of the molecules in the solution is unstable. The peptide chain folding modes are diverse and unstable, and there are often phenomena of sometimes dissolving clearly and sometimes precipitating. In the purification method of tirzepatide provided by the present invention, there is a double salt in the buffer solvent, which greatly improves the ionization intensity of tirzepatide, making its spatial form stable and single, exposing its hydrophilic groups on the outer side of the molecule, and greatly increasing its water solubility. The concentration of the dissolved crude product is high, and the sample can be stably placed for one week without precipitation; moreover, this buffer solvent can be easily removed during the subsequent salt transfer process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following further elaborates on the technical solutions of the present invention with specific examples in the accompanying drawings, but does not constitute any limitation to the present invention.

[0018] Figure 1 This is the HPLC chromatogram of the crude exenatide in the embodiments of the present invention; Figure 2 This is the HPLC chromatogram of the purified exenatide in Example 1 of the present invention; Figure 3 This is the MS chromatogram of the purified exenatide in Example 1 of the present invention. Detailed implementation manners

[0019] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] Example 1. The X reagent consists of DMSO, DMF, diammonium hydrogen phosphate, citric acid and ammonium bicarbonate; the Y reagent consists of acetonitrile, methanol and tetrahydrofuran; the buffer solvent is obtained by mixing X and Y in a volume ratio of 95:5. Weigh 112.08 g of the crude exenatide and slowly add it to 2600 mL of the buffer solvent and stir to dissolve; after the crude exenatide is dissolved, stir at 40 °C for 4 h; filter through a 0.45 μm microporous membrane to obtain the crude exenatide solution. Observe the appropriate amount of the crude exenatide solution. After one week, observe the state of the solution and find that the solution is still clear and no precipitation occurs.

[0021] Analyze and detect the crude exenatide solution. The crude exenatide solution is loaded onto an RP-HPLC (column length 150 mm), and the loading amount is 15 g; among them, the stationary phase is selected as Daisogel SP-100-8-C8-PK. Quantify 59.07 g with the standard product, and the injection volume per needle is 650 mL; perform HPLC purification and preparation, and collect the fractions with a purity greater than 98%. The instrument used is the LC1000 preparative liquid chromatograph of Beijing Aonuo Technology Co., Ltd., equipped with a UV detector, and the 100DAC of Beijing Aonuo Technology Co., Ltd.

[0022] The chromatographic conditions for HPLC purification are: Chromatographic column: C8 column, column length: 100 Í 150 mm (packing Daisogel SP-100-8-C8-PK); Mobile phase A is: buffer solvent; Mobile phase B is: Mobile phase A: pure acetonitrile = 30:70; Time and mobile phase gradient: The preparation gradient B% is 30% - 38%, and the preparation time is 60 min; Flow rate: 250 mL / min; Detection wavelength: 220 nm; Sample size: 15 g.

[0023] Fractions with a purity greater than 98% were combined and an equal volume of purified water was added to obtain the solution of teduglutide to be salt-converted. The solution of teduglutide to be salt-converted was salt-converted and prepared according to the following method: Instrument: LC1000 preparative liquid chromatograph of Beijing Aonuo Technology Co., Ltd., equipped with an ultraviolet detector Preparation column: C8 column, column length: 100×150 mm (packing particle size 8 μm, pore size 100 Å) Mobile phase: A 0.3% aqueous acetic acid solution, B acetonitrile, C 50 mM ammonium acetate Time and mobile phase gradient: Salt conversion: B% is 5%, C% is 95%. Preparation time is 14 min; Elution: B% is 5%, A% is 95%, preparation time is 14 min; B% is 70%, A% is 30%, gradient time is 50 min; Flow rate: 250 mL / min; Detection wavelength: 220 nm; Sample size: 32.5 g (calculated based on the pure peptide of teduglutide).

[0024] After salt-conversion preparation, it was concentrated and freeze-dried to obtain 50.56 g of teduglutide pure peptide with a purity of 99.27%.

[0025] Example 2, the chromatographic conditions for HPLC purification and salt-conversion preparation are the same as those in Example 1.

[0026] Reagent X consists of DMSO, citric acid and ammonium bicarbonate; Reagent Y consists of acetonitrile and methanol; X:Y is mixed at a volume ratio of 95:5 to obtain a buffer solvent. Weigh 380.67 g of crude teduglutide and slowly add it to 9500 mL of the buffer solvent and stir to dissolve; after dissolution, stir at 40 °C for 4 h. Filter through a 0.45 μm microporous membrane to obtain a solution of crude teduglutide; analyze and detect the solution of crude teduglutide, quantitatively determine 204.81 g with a standard product, and the sample injection volume per needle is 680 mL; perform HPLC purification preparation and collect fractions with a purity greater than 98%. Fractions with a purity greater than 98% were combined and an equal volume of purified water was added to obtain the solution of teduglutide to be salt-converted. The solution of teduglutide to be salt-converted was salt-converted and prepared, and after salt conversion, it was concentrated and freeze-dried to obtain 181.47 g of teduglutide pure peptide with a purity of 99.39%.

[0027] Example 3, the chromatographic conditions for HPLC purification and salt-conversion preparation are the same as those in Example 1.

[0028] Reagent X consists of DMF, HAC, phosphoric acid, ammonia water, triethylamine, sodium hydroxide, H2O, diammonium hydrogen phosphate and Tris; Reagent Y consists of acetonitrile, methanol, isopropanol and tetrahydrofuran; Buffer solvent is obtained by mixing X:Y at a volume ratio of 95:5. Weigh 368.15 g of crude teduglutide and slowly add it to 9200 mL of buffer solvent and stir to dissolve; After dissolution, stir at 45 °C for 5 h. Filter through a 0.45 μm microporous membrane to obtain a crude teduglutide solution. Analyze and detect the crude teduglutide solution, quantify 182.31 g with a standard product, and the sample injection volume per needle is 760 mL; Conduct HPLC purification and preparation, and collect fractions with a purity greater than 98%. Combine the fractions with a purity greater than 98% and add an equal volume of purified water to obtain the teduglutide solution to be salt-converted. Prepare the salt conversion of the teduglutide solution to be salt-converted; After salt conversion, concentrate and freeze-dry to obtain 151.61 g of refined teduglutide with a purity of 99.33%.

[0029] Example 4, the column length is 100 nm, and other purification and preparation conditions remain unchanged.

[0030] Reagent X consists of DMSO, citric acid and ammonium bicarbonate; Reagent Y consists of acetonitrile and methanol; Buffer solvent is obtained by mixing X:Y at a volume ratio of 95:5. Weigh 120.25 g of crude teduglutide and slowly add it to 3000 mL of buffer solvents X and Y and stir to dissolve; After the sample is dissolved, stir at 35 °C for 5 h. Filter through a 0.45 μm microporous membrane to obtain a crude teduglutide solution; Analyze and detect the crude teduglutide solution, quantify 64.94 g with a standard product, and the sample injection volume per needle is 600 mL; Conduct HPLC purification and preparation, and collect the teduglutide fractions with a purity greater than 98%.

[0031] Combine the fractions with a purity greater than 98% and add an equal volume of purified water to obtain the teduglutide solution to be salt-converted. Prepare the salt conversion of the teduglutide solution to be salt-converted, and after salt conversion, concentrate and freeze-dry to obtain 22.47 g of refined teduglutide with a purity of 98.51%.

[0032] Example 5, the column length is 100 nm, and other purification and preparation conditions remain unchanged.

[0033] Reagent X consists of DMSO, citric acid and ammonium bicarbonate; Reagent Y consists of acetonitrile and methanol; Buffer solvent is obtained by mixing X:Y at a volume ratio of 95:5. Weigh 115.22 g of crude teduglutide and slowly add it to 2880 mL of buffer solvents X and Y and stir to dissolve; After the sample is dissolved, stir at 35 °C for 5 h. Filter through a 0.45 μm microporous membrane to obtain a crude teduglutide solution; Analyze and detect the crude teduglutide solution, quantify 56.99 g with a standard product, and the sample injection volume per needle is 600 mL; Conduct HPLC purification and preparation, and collect the teduglutide fractions with a purity greater than 98%.

[0034] The fractions with a purity greater than 98% were combined, and an equal volume of purified water was added to obtain the solution of tildutide to be salt-converted. The solution of tildutide to be salt-converted was subjected to salt conversion preparation, and after salt conversion, it was concentrated and freeze-dried to obtain 22.80 g of tildutide fine peptide with a purity of 98.22%.

[0035] Example 6, the length of the chromatographic column was 250 mm, and the other conditions for HPLC purification were the same as those in Example 1. The chromatographic conditions for salt conversion preparation were as follows: Instrument: LC1000 preparative liquid chromatograph of Beijing Aonuo Technology Co., Ltd., equipped with an ultraviolet detector; Chromatographic column: C8 column, column length: 100×250 mm (packing particle size 8 μm, pore size 100 Å); Mobile phase: A 0.3% acetic acid aqueous solution, B acetonitrile, C 50 mM ammonium acetate; Time and mobile phase gradient: Salt conversion: B% was 5%, C% was 95%. Preparation time was 23 min Elution: B% was 5%, A% was 95%, preparation time was 23 min; B% was 70%, A% was 30%, preparation time was 50 min; Flow rate: 250 mL / min; Detection wavelength: 220 nm; Sample injection volume: 32.5 g (calculated as tildutide fine peptide).

[0036] Reagent X was composed of diammonium hydrogen phosphate, Tris, citric acid and ammonium bicarbonate; reagent Y was composed of methanol and isopropanol; X:Y was mixed in a volume ratio of 95:5 to obtain a buffer solution. 123.05 g of crude tildutide was weighed and slowly added to 3000 mL of buffer solvent and stirred to dissolve; after dissolution, it was stirred at 40 °C for 4 h; it was filtered through a 0.45 μm microporous membrane to obtain a crude tildutide solution. The crude tildutide solution was analyzed and detected, 66.45 g was quantified with a standard product, the sample injection volume per needle was 600 mL, and HPLC purification was carried out, and the fractions with a purity greater than 98% were collected. The fractions with a purity greater than 98% were combined, and an equal volume of purified water was added to obtain the solution of tildutide to be salt-converted. The solution of tildutide to be salt-converted was subjected to salt conversion preparation, and after salt conversion, it was concentrated and freeze-dried to obtain 53.16 g of tildutide fine peptide with a purity of 99.01%.

[0037] Example 7, the chromatographic conditions for HPLC purification and salt conversion preparation were the same as those in Example 6.

[0038] Reagent X consists of diammonium hydrogen phosphate, Tris, citric acid and ammonium bicarbonate; Reagent Y consists of methanol and isopropanol; X and Y are mixed at a volume ratio of 95:5 to obtain a buffer solution. Weigh 119.58 g of the crude product of tirzepatide and slowly add it to 3000 mL of the buffer solvent, stirring to dissolve; after dissolution, stir at 40 °C for 4 h; filter through a 0.45 μm microporous membrane to obtain the crude tirzepatide solution. Analyze and detect the crude tirzepatide solution, quantitatively analyze 64.57 g with a standard product, with a sample injection volume of 600 mL per injection, and perform HPLC purification to collect fractions with a purity greater than 98%. Combine the fractions with a purity greater than 98% and add an equal volume of purified water to obtain the tirzepatide solution to be salt-converted. Prepare the salt conversion of the tirzepatide solution to be salt-converted, concentrate and freeze-dry after salt conversion to obtain 53.00 g of the purified peptide of tirzepatide with a purity of 98.69%.

[0039] The experimental data of Examples 1 to 7 are shown in Table 1.

[0040] Table 1

[0041] In summary, according to the purification method of tirzepatide of the present invention, when dissolving the crude peptide, the dissolution ability of the crude peptide is greatly improved, the concentration of the crude product can reach about 40 mg / mL, and the sample remains stable and does not precipitate after being placed for one week. The purification and salt conversion preparation steps are simple, without other complex processes, which is convenient for large-scale production in the later stage; it can reduce the purification cost and improve the purity.

[0042] When the chromatographic length is between 100 and 250 nm, using the purification method of the present invention, the purity of the purified peptide of tirzepatide finally obtained all reaches more than 98%; when the chromatographic length is 150 nm, the purification effect is the best, and the purity of the purified peptide of tirzepatide can all reach more than 99%; when the chromatographic length is 250 nm, the purity of the purified peptide of tirzepatide can reach about 99%; in some embodiments, a product with a purity of 99% can be obtained by one-step purification, greatly shortening the purification cycle.

[0043] The above is the preferred embodiment of the present invention and is not limited to the present invention. For those skilled in the art, several improvements and variations made without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for purifying teduglutide, characterized in that, It includes the following steps: (1) Dissolve the crude teduglutide in a buffer solvent, stir at 35 - 45 °C after dissolution, and then filter to obtain a crude teduglutide solution; (2) Purify the crude teduglutide solution by HPLC and collect the fractions with a purity greater than 98%; (3) Add an equal volume of purified water to the fractions, carry out salt conversion, concentration, and lyophilization to obtain the purified teduglutide; Among them, the buffer solvent is composed of X and Y mixed in a volume ratio of 90 - 95:5 - 10; X is selected from one or more of DMSO, DMF, HAC, phosphoric acid, ammonia water, triethylamine, sodium hydroxide, H2O, diammonium hydrogen phosphate, Tris, citric acid, and ammonium bicarbonate; Y is selected from one or more of acetonitrile, methanol, isopropanol, and tetrahydrofuran.

2. The purification method of tirzepatide according to claim 1, wherein In the step (1), the volume ratio of the buffer solvents X and Y is 95:

5.

3. The purification method of tirzepatide according to claim 1, wherein In the step (1), the stirring time is 3 - 5 hours.

4. The purification method of tildrakizumab according to claim 1, wherein In the step (2), the chromatographic conditions for HPLC purification are: Chromatographic column: C8 column, column length: 100 - 250 mm; Mobile phase A: buffer solvent; Mobile phase B: a mixture of mobile phase A and pure acetonitrile with a volume ratio of 30:70; Time and mobile phase gradient: The preparation gradient B% is 30% - 38%, and the preparation time is 60 min; Flow rate: 250 mL / min; Injection volume: 15 g.

5. The purification method of tirzepatide according to claim 1 or 4, characterized in that, In the step (2), the column length of the chromatographic column during HPLC purification is 150 nm.

6. The purification method of tildrakizumab according to claim 1 or 4, characterized in that, The detector used during the HPLC purification is an ultraviolet detector, and the detection wavelength is 220 nm.

7. The purification method of tirzepatide according to claim 1, wherein, The chromatographic conditions for salt conversion in the step (3) are: Chromatographic column: C8 column, column length: 100 - 250 mm; Mobile phase: A 0.3% aqueous acetic acid solution, B acetonitrile, C 50 mM ammonium acetate; Time and mobile phase gradient: Salt conversion: B% is 5%, C% is 95%; the preparation time is 14 - 23 min; Elution: B% is 5%, A% is 95%; the preparation time is 14 - 23 min; B% is 70%, A% is 30%, and the gradient time is 50 min; Flow rate: 250 mL / min; Injection volume: 32.5 g.

8. The purification method of tildrakizumab according to claim 7, wherein The preparation time for both salt conversion and elution is 14 min.

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