A method for purifying Retatrutide

Abstract

The present invention is a purification method of Retatrutide, belonging to the technical field of pharmaceutical chemistry; the method comprises dissolving crude Retatrutide peptide in purified water, adjusting the pH to weak alkalinity with ammonia water, filtering to obtain a crude peptide solution after complete dissolution; taking the crude peptide solution, purifying it through a C18 hybrid column, gradient eluting with a mixed solution of solution A and solution B, collecting the eluted fractions, and obtaining pure Retatrutide; the solution A is an aqueous solution of ammonium dihydrogen phosphate; the solution B is a mixed solution of acetonitrile and methanol. The method of the present invention optimizes multi-step purification into one-step purification, spray drying replaces traditional freeze drying, omits the concentration step, saves time, is simple to operate, improves the purification yield, and is more conducive to the industrialized large-scale production of Retatrutide.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a method for purifying Retatrutide. Background Art

[0002] Retatrutide is a triple receptor agonist of glucose-dependent insulinogenic polypeptide (GIP), glucagon-like peptide 1 (GLP-1) and glucagon (GCG). Retatrutide can lower blood sugar, lose weight, reduce the risk of cardiovascular events, and even has a good effect on non-alcoholic hepatitis.

[0003] Retatrutide has the following structure: Tyr 1 -Aib-Gln-Gly-Thr 5 -Phe-Thr-Ser-Asp-Tyr 10 -Ser-Ile-(α)MeLeu-Leu-Asp 15 -Lys-Lys(Eicosanedioicacid-γ-Glu-AEEA)-Ala-Gln-Aib 20 -Ala-Phe-Ile-Glu-Tyr 25 -Leu-Leu-Glu-Gly-Gly 30 -Pro-Ser-Ser-Gly-Ala 35 -Pro-Pro-Pro-Ser 39 -NH2.

[0004] At present, there are many public technologies for polypeptide purification, such as CN201811601939.8 discloses a method for purifying triptorelin, CN201510004136.4 discloses a method for purifying and preparing triptorelin, CN201811407810.3 and CN201811603717.X disclose a method for separating and purifying liraglutide, and CN110845602A discloses a method for separating and purifying semaglutide. However, due to the sequence differences of different polypeptides, the above methods are not suitable for the separation and purification of Retatrutide. In particular, since the peptide chain of Retatrutide is longer, the side chain contains longer modifications, and the peptide sequence contains amino acids such as Ser that are easily isomerized during the synthesis process, resulting in isomeric impurities in the crude peptide, there are more missing impurities and isomeric impurities generated during the synthesis, and the crude Retatrutide peptide needs to be purified. However, the yield of the retatrutide purification method disclosed in the prior art is relatively low. For example, CN118754966A discloses a method of using octadecylsilane bonded silica gel as filler and trifluoroacetic acid aqueous solution as eluent to purify the solution containing retatrutide (crude peptide purity 76.3%) by reverse phase chromatography for the first purification and the second purification, and finally obtains a refined peptide with a purity of 99.5%. Although this method also improves the purity of the peptide, it involves multiple purification steps and is relatively cumbersome.

[0005] CN117024528A discloses that a first purification is performed using C8 filler, ammonium acetate / water solution as phase A, and acetonitrile as phase B, and an acetic acid aqueous solution as phase A and an acetic acid acetonitrile solution as phase B, and finally a refined peptide with a purity greater than 99.5% is obtained, but the final purification yield of the process is only 57.9%. In view of this, how to optimize the purification process of Retatrutide has become a difficulty in the preparation process of Retatrutide and one of the bottlenecks in realizing the large-scale industrial production of Retatrutide. Therefore, it is necessary to explore a Retatrutide purification method with simpler operation, which can both ensure the purity of the product and improve the yield of Retatrutide. Summary of the invention

[0006] The purpose of the present invention is to provide a novel purification method of Retatrutide in view of the deficiencies of the prior art. The purification method of the present invention is simple to operate, only requires one purification step to achieve a high yield, and is more conducive to the industrialized scale-up production of Retatrutide.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solution: The present invention is a method for purifying Retatrutide, which is characterized in that the steps are as follows: Step 1: Dissolve the crude peptide Retatrutide in purified water, adjust the pH to 8.0-8.5 with aqueous ammonia, and filter after complete dissolution to obtain a crude peptide solution; Step 2: taking the crude peptide solution, purifying it through a C18 hybrid column, performing gradient elution with a mixed solution of solution A and solution B, collecting the eluted fractions, and obtaining pure Retatrutide; The solution A is a 40-50 mmol / L aqueous solution of ammonium dihydrogen phosphate, and the pH value is adjusted to 8.0-8.5 with ammonia water; The B solution is 45%-55% acetonitrile + 55%-45% methanol solution; The volume fraction ratio of solution A to solution B is 73%:27%→37%:63%, and the gradient elution time is 63min-72min; Step 3: spray-dry the purified product to obtain the finished product of Retatrutide.

[0008] The purification method of Retatrutide of the present invention, its further preferred technical scheme is: 1. In step 1, the purification chromatographic column is a C18 hybrid silica gel column.

[0009] 2. In step 1, the volume concentration of ammonia water used for adjusting pH is 10%-20%.

[0010] 3. In step 2, solution A is a 40-50 mmol / L aqueous solution of ammonium dihydrogen phosphate, and the pH is adjusted to 8.0-8.5 with aqueous ammonia, and solution B is a 45%-55% acetonitrile + 55%-45% methanol solution.

[0011] 4. In step 2, the purification flow rate is 50 ml / min.

[0012] 5. In step 3, the spray drying temperature is 75°C-85°C.

[0013] 6. In step 1, crude Retatrutide peptide is prepared by solid phase synthesis, and the purity of crude Retatrutide peptide is 65%-85%.

[0014] 7. In step 2: Preferred purification conditions are as follows: Chromatographic column: a chromatographic column with hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm; mobile phase: Phase A: 40mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 8.0 with ammonia water; Phase B: 45% acetonitrile + 55% methanol solution; Flow rate: 50mL / min; Detection wavelength: 220nm; Gradient: B%: 27%→63%, 63 min; The purification process is as follows: the chromatographic column is equilibrated with 10% B phase and then loaded with sample; after loading, elution is performed, and the linear gradient elution is performed for 63 minutes; the target peak is collected to obtain a fraction with a purity greater than 99.0%, and the collected qualified fraction is spray-dried at a temperature of 75°C to obtain the finished Retatrutide product.

[0015] 8. In step 2: Preferred purification conditions are: Chromatographic column: a chromatographic column with hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm; mobile phase: Phase A: 45mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 8.3 with ammonia water; Phase B: 50% acetonitrile + 50% methanol solution; Flow rate: 50mL / min; Detection wavelength: 220nm; Gradient: B%: 27%→63%, 67 min; The purification process is: The chromatographic column was equilibrated with 10% B phase and then loaded with the sample; after loading, elution was performed with a linear gradient elution for 67 minutes. The target peak was collected to obtain a fraction with a purity greater than 99.0%, and the collected qualified fraction was spray-dried at a temperature of 80°C to obtain the finished product of Retatrutide.

[0016] 9. In step 2: Preferred purification conditions are: Chromatographic column: a chromatographic column with hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm; mobile phase: Phase A: 50mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 8.5 with ammonia water; Phase B: 55% acetonitrile + 45% methanol solution; Flow rate: 50mL / min; Detection wavelength: 220nm; Gradient: B%: 27%→63%, 72 min; Purification process: The chromatographic column was equilibrated with 10% B phase and then loaded with sample; after loading, elution was performed, and the linear gradient elution was performed for 72 minutes; the target peak was collected to obtain a fraction with a purity greater than 99.0%, and the collected qualified fraction was spray-dried at a temperature of 85° C. to obtain the finished Retatrutide product.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The method of the present invention optimizes multi-step purification into one-step purification, spray drying replaces traditional freeze drying, omits the concentration step, saves time, is simple to operate, improves the purification yield, and is more conducive to the industrialized large-scale production of Retatrutide. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described below in conjunction with the embodiments. The reagents and raw materials used in the following purification methods can all be purchased from the market.

[0019] Example 1: Purification of crude Retatrutide peptide Retatrutide was synthesized in solid phase using the existing technology, and the purity of the crude peptide was 72%.

[0020] The crude peptide with a purity of 72% was purified by the following method: Sample treatment: Dissolve the solid crude peptide containing 9.9 g of the target peptide in purified water, adjust the pH to 8.0 with 10% ammonia aqueous solution, stir to completely dissolve the sample, filter with a 0.45 um filter membrane, and collect the filtrate for later use.

[0021] Purification conditions: Chromatographic column: a chromatographic column with C18 hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm. Mobile phase: Phase A: 40 mmol / L ammonium dihydrogen phosphate aqueous solution, pH = 8.0 adjusted with ammonia water; Phase B: 45% acetonitrile + 55% methanol solution, flow rate: 50 mL / min, detection wavelength: 220 nm. Gradient: B%: 27% → 63%, 63 min.

[0022] Purification process: The chromatographic column was equilibrated with 10% phase B and then loaded with 2.5 g of sample. After loading, elution was performed with a linear gradient elution of 63 min. The target peak was collected to obtain a fraction with a purity greater than 99.0%. The collected qualified fractions were spray-dried at a temperature of 75°C to obtain 9.1 g of Retatrutide with a purity of 99.15%, and the purification yield was 91.9%.

[0023] Example 2: Purification of crude Retatrutide peptide Retatrutide was synthesized in solid phase using the existing technology, and the purity of the crude peptide was 74%.

[0024] The crude peptide with a purity of 74% was purified by the following method: Sample treatment: Dissolve the solid crude peptide containing 10.2g of the target peptide in pure water, adjust the pH to 8.3 with 15% ammonia aqueous solution, stir to completely dissolve the sample, filter with a 0.45um filter membrane, and collect the filtrate for later use.

[0025] Purification conditions: Chromatographic column: a chromatographic column with C18 hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm. Mobile phase: Phase A: 45 mmol / L ammonium dihydrogen phosphate aqueous solution, pH = 8.3 adjusted with ammonia water; Phase B: 50% acetonitrile + 50% methanol solution, flow rate: 50 mL / min, detection wavelength: 220 nm. Gradient: B%: 27% → 63%, 67 min.

[0026] Purification process: The chromatographic column was equilibrated with 10% B phase and then loaded with sample, the loading amount was 2.5g. After loading, elution was performed, and the linear gradient elution was 67min. The target peak was collected to obtain the fraction with a purity greater than 99.0%. The qualified fractions were collected and used as 80 ℃ The reaction mixture was spray dried at a temperature of 1000 ℃ to obtain 9.4 g of Retatrutide with a purity of 99.28% and a purification yield of 92.2%.

[0027] Example 3: Purification of crude Retatrutide peptide Retatrutide was synthesized in solid phase using the existing technology, and the purity of the crude peptide was 70%.

[0028] The crude peptide with a purity of 70% was purified by the following method: Sample treatment: Dissolve the solid crude peptide containing 12.5g of the target peptide in pure water, adjust the pH to 8.5 with 20% ammonia aqueous solution, stir to completely dissolve the sample, filter with a 0.45um filter membrane, and collect the filtrate for later use.

[0029] Purification conditions: Chromatographic column: a chromatographic column with hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm. Mobile phase: Phase A: 50 mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 8.5 with ammonia water; Phase B: 55% acetonitrile + 45% methanol solution, flow rate: 50 mL / min, detection wavelength: 220 nm. Gradient: B%: 27% → 63%, 72 min.

[0030] Purification process: The chromatographic column was equilibrated with 10% phase B and then loaded with 2.5 g of sample. After loading, elution was performed with a linear gradient elution of 72 min. The target peak was collected to obtain a fraction with a purity greater than 99.0%. The collected qualified fraction was spray-dried at a temperature of 85°C to obtain 11.6 g of Retatrutide with a purity of 99.21%, and the purification yield was 92.8%.

[0031] In the above examples, the detection and calculation methods of yield and purity all adopt conventional methods.

[0032] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for purifying Retatrutide, characterized in that: The steps are as follows: Step 1: Dissolve the crude peptide Retatrutide in purified water, adjust the pH to 8.0-8.5 with aqueous ammonia, and filter after complete dissolution to obtain a crude peptide solution; Step 2: taking the crude peptide solution, purifying it through a C18 hybrid column, performing gradient elution with a mixed solution of solution A and solution B, collecting the eluted fractions, and obtaining pure Retatrutide; The solution A is a 40mmol / L-50mmol / L aqueous solution of ammonium dihydrogen phosphate, and the pH value is adjusted to 8.0-8.5 with ammonia water; The B solution is 45%-55% acetonitrile + 55%-45% methanol solution; The volume fraction ratio of solution A to solution B is 73%:27%→37%:63%, and the gradient elution time is 63min-72min; Step 3: spray-dry the purified product to obtain the finished product of Retatrutide.

2. The method for purifying Retatrutide according to claim 1, characterized in that: In step 1, the chromatographic column used for purification is a C18 hybrid silica gel column.

3. The method for purifying Retatrutide according to claim 1, characterized in that: In step 1, the volume concentration of ammonia water used for adjusting pH is 10%-20%.

4. The method for purifying Retatrutide according to claim 1, characterized in that: In step 2, solution A is a 40mmol / L-50mmol / L aqueous solution of ammonium dihydrogen phosphate, with ammonia water adjusting the pH to 8.0-8.5, and solution B is a 45%-55% acetonitrile + 55%-45% methanol solution.

5. The method for purifying Retatrutide according to claim 1, characterized in that: In step 2, the flow rate of purification was 50 ml / min.

6. The method for purifying Retatrutide according to claim 1, characterized in that: In step 3, the spray drying temperature is 75°C-85°C.

7. The method for purifying Retatrutide according to claim 1, characterized in that: In step 1, crude Retatrutide peptide is prepared by solid phase synthesis, and the purity of crude Retatrutide peptide is 65%-85%.

8. The method for purifying Retatrutide according to claim 1, characterized in that: In step 2: The purification conditions were as follows: Chromatographic column: Chromatographic column with C18 hybrid silica gel as stationary phase, column diameter and length: 50mm×270mm; Mobile phase: Phase A: 40mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 8.0 with ammonia water; Phase B: 45% acetonitrile + 55% methanol solution; Flow rate: 50mL / min; Detection wavelength: 220nm; Gradient: B%: 27%→63%, 63 min; The purification process is as follows: the column is equilibrated with 10% phase B and then loaded with the sample; After loading, elution was performed with a linear gradient elution of 63 min; The target peak was collected to obtain a fraction with a purity greater than 99.0%, and the qualified fraction was spray-dried at a temperature of 75° C. to obtain the finished product of Retatrutide.

9. The method for purifying Retatrutide according to claim 1, characterized in that: In step 2: The purification conditions are: Chromatographic column: A chromatographic column with C18 hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm; Mobile Phase: Phase A: 45mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 8.3 with ammonia water; Phase B: 50% acetonitrile + 50% methanol solution; Flow rate: 50mL / min; Detection wavelength: 220nm; Gradient: B%: 27%→63%, 67 min; The purification process is: The column was equilibrated with 10% phase B and then loaded with the sample; After loading, elution was performed with a linear gradient elution of 67 minutes. The target peak was collected to obtain a fraction with a purity greater than 99.0%, and the collected qualified fraction was spray-dried at a temperature of 80°C to obtain the finished product of Retatrutide.

10. The method for purifying Retatrutide according to claim 1, characterized in that: In step 2: The purification conditions are: Chromatographic column: A chromatographic column with C18 hybrid silica gel as the stationary phase, with a column diameter and length of 50 mm × 270 mm; Mobile Phase: Phase A: 50mmol / L ammonium dihydrogen phosphate aqueous solution, pH adjusted to 8.5 with ammonia water; Phase B: 55% acetonitrile + 45% methanol solution; Flow rate: 50mL / min; Detection wavelength: 220nm; Gradient: B%: 27%→63%, 72 min; Purification process: The column was equilibrated with 10% phase B and then loaded with the sample; After loading, elution was performed with a linear gradient elution of 72 min; The target peak was collected to obtain a fraction with a purity greater than 99.0%, and the qualified fraction was spray-dried at a temperature of 85° C. to obtain the finished product of Retatrutide.