Smeglutide precursor purification method

Abstract

The invention provides a method for purifying a semeglutide precursor, and belongs to the technical field of polypeptide preparation.The method comprises the steps that a precursor crude sample solution obtained after enzyme digestion in the synthesis process is treated through isoelectric point precipitation, precipitation is taken and suspended again, then hydrophobic chromatography purification is conducted, and the purified semeglutide precursor is obtained. Meanwhile, the hydrophobic chromatography process is easy for linear amplification and low in production cost, and the feasibility of amplified production is greatly improved; besides, most of impurities such as enzymes and pigments can be removed after pretreatment by an isoelectric precipitation process, and after hydrophobic chromatography purification, the purity of the semeglutide precursor is greatly improved and can reach 95% or above, the hydrophobic purification yield is greater than 80%, product related impurities generated by enzyme digestion are effectively removed, and the product quality is improved. The purified precursor can be directly used for subsequent modification and other process steps.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide preparation, and particularly relates to a method for purifying a semaglutide precursor. Background Art

[0002] Semaglutide is a new long-acting GLP-1 analog with the following peptide sequence: His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys20(AEEA-AEEAγ-Glu-Octadecanedioic)-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH. From the peptide sequence, the amino acid at position 2 is a non-natural α-aminoisobutyric acid (Aib), and the side chain of the amino acid lysine at position 20 of the peptide sequence contains an octadecanoic acid fatty chain modified with polyethylene glycol (PEG), which greatly enhances the hydrophilicity of the entire peptide. It can not only bind tightly to albumin and resist degradation by dipeptidyl peptidase 4 (DPP-4), but also reduce renal excretion and significantly prolong the half-life, making it suitable for subcutaneous injection once a week to achieve a long circulation effect.

[0003] At present, the synthesis of semaglutide is mainly achieved by gradually coupling each amino acid unit to extend the peptide chain, such as a method for synthesizing semaglutide provided by the invention patent with publication number CN116120427A. However, during the synthesis process, many missing peptide impurities with properties close to the main component will be generated, for example, missing peptide impurities caused by the loss of simple structure and small molecular weight amino acids such as alanine (Ala) or glycine (Gly), especially the 18th and 19th amino acids of the peptide chain are two consecutive Alas, which makes it easier for a single Ala to be lost and produce impurities, which has a greater impact on subsequent modification processes. Summary of the invention

[0004] Based on the above problems in the prior art, the present invention provides a method for purifying a semaglutide precursor, which first uses isoelectric precipitation to treat a crude precursor sample solution obtained after enzyme cleavage during the synthesis process, resuspends the precipitate, and then purifies it by hydrophobic chromatography to obtain a purified semaglutide precursor, comprising the following detailed steps:

[0005] Step S1, taking a crude sample solution of semaglutide precursor obtained after enzyme cleavage, adjusting the pH value of the crude sample solution to 4.8-5.0, allowing the solution to stand for precipitation, discarding the supernatant and collecting the precipitate, adding water to the precipitate to resuspend it, and adjusting the pH value of the resuspended solution to 7.0-9.0 to redissolve the precipitate to obtain a reconstituted solution;

[0006] Step S2, using a chromatography column to separate and purify the complex solution obtained in step S1, the filler of the chromatography column is Generik MC60-HIC Butyl, and the chromatography treatment conditions are:

[0007] Equilibration: Equilibrate the column with 2CV equilibration buffer;

[0008] Sampling: Take the reconstituted solution and load it. The loading volume should not exceed 10g / L.

[0009] Washing: Wash with 2.5CV equilibration buffer;

[0010] Washing: Use 4CV washing buffer to clean impurities;

[0011] Elution: isocratic elution with 2.5CV pure water, collecting the elution liquid corresponding to the main peak to obtain the purified semaglutide precursor.

[0012] Preferably, in step S1, the pH value of the crude sample solution is adjusted to 4.9, the pH value of the resuspension solution is adjusted to 8.0, and the static precipitation time is greater than 20 minutes.

[0013] Preferably, in step S1, the pH value is adjusted using an inorganic acid or an inorganic base, the inorganic acid includes one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and sodium dihydrogen phosphate, and the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonia water, calcium hydroxide, and sodium phosphate.

[0014] Preferably, the equilibrium buffer is 450 mM NaCl + 45% to 55% water with a pH value of 7.8 to 8.8.

[0015] Preferably, the equilibration buffer is 450 mM NaCl + 50% water with a pH value of 8.3.

[0016] Preferably, the washing buffer is 450 mM NaCl + 80% to 90% water with a pH value of 7.8 to 8.8.

[0017] Preferably, the washing buffer is 450 mM NaCl + 85% water with a pH value of 8.3.

[0018] Preferably, the method for purifying a semaglutide precursor further comprises step S3, regenerating the chromatography column filler: regenerating with 2CV 0.5M NaOH and washing with 2CV pure water.

[0019] The beneficial effects of the present invention are:

[0020] 1. The crude sample solution of semaglutide precursor was pretreated by isoelectric precipitation process, and then the semaglutide precursor was purified by hydrophobic chromatography process. The precipitation process is simple and easy to operate, and the hydrophobic chromatography process is easy to linearly scale up and has low production cost, which greatly improves the feasibility of scale-up production.

[0021] 2. After pretreatment by isoelectric precipitation process, most impurities such as enzymes and pigments can be removed. After purification by hydrophobic chromatography, the purity of semaglutide precursor is greatly improved, the purity can reach more than 95%, and the hydrophobic purification yield is greater than 80%, which effectively removes product-related impurities generated by enzyme cleavage. The purified precursor can be directly used in subsequent modification and other process steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a comparison of the electrophoresis of semaglutide precursor before and after purification, with the left side showing after purification and the right side showing before purification. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is described below in conjunction with the accompanying drawings and specific embodiments.

[0024] This embodiment provides a method for purifying a semaglutide precursor, which first uses isoelectric precipitation to treat a crude precursor sample solution obtained after enzyme cleavage during the synthesis process, resuspends the precipitate, and then purifies it by hydrophobic chromatography to obtain a purified semaglutide precursor, including the following detailed steps:

[0025] Step S1, taking the crude sample solution of semaglutide precursor obtained after enzyme cleavage, adjusting the pH value of the crude sample solution to 4.9, standing and precipitating for 30 minutes, discarding the supernatant and collecting the precipitate, adding water to the precipitate to resuspend it, and adjusting the pH value of the resuspension to 8.0 to redissolve the precipitate to obtain a resolubilized solution. In this step, an inorganic acid or an inorganic base is used to adjust the pH value. The inorganic acid includes one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and sodium dihydrogen phosphate. In this embodiment, hydrochloric acid is selected; the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonia water, calcium hydroxide, and sodium phosphate. In this embodiment, sodium carbonate is selected;

[0026] Step S2, using a chromatography column to separate and purify the complex solution obtained in step S1, the filler of the chromatography column is Generik MC60-HIC Butyl, and the chromatography treatment conditions are:

[0027] Balance: Use 2CV of balance buffer to balance the column until the pH and conductivity of the effluent are consistent with the influent values. After the balance is completed, the sample can be loaded. The balance buffer is 450mM NaCl + 50% water with a pH value of 8.3 and a flow rate of 42.21cm / h;

[0028] Loading: Add the reconstituted solution to the chromatography column. The loading volume should not exceed 10 g / L and the flow rate should be 42.21 cm / h.

[0029] Flushing: Flushing with 2.5CV equilibration buffer at a flow rate of 42.21cm / h, wherein the equilibration buffer is 450mM NaCl+50% water with a pH value of 8.3;

[0030] Washing: Use 4CV washing buffer to wash impurities at a flow rate of 42.21 cm / h. The washing buffer is 450 mM NaCl + 85% water with a pH value of 8.3;

[0031] Elution: use 2.5CV pure water isocratic elution at a flow rate of 42.21cm / h, collect the elution liquid corresponding to the main peak, and obtain the purified semaglutide precursor;

[0032] Step S3, regeneration of the chromatography column filler: regeneration with 2CV 0.5M NaOH, and then rinse with 2CV pure water, and the filler is recovered for standby use or storage. The flow rate of regeneration and rinse is 42.21cm / h.

[0033] The purified semaglutide precursor eluted in step S2 was subjected to SDS-PAGE detection, and the crude precursor sample solution before purification was used as a control. The results are as follows: Figure 1 As shown, the content of impurity proteins in the semaglutide precursor purified by the purification method of the present invention is greatly reduced, and a semaglutide precursor with higher purity is obtained.

[0034] The purified semaglutide precursor eluted in step S2 was then subjected to LC-MS-MS quantitative analysis. The results showed that the purity could reach more than 95%, the hydrophobic purification yield was greater than 80%, and the product-related impurities generated by enzymatic cleavage were effectively removed. The purified precursor can be directly used in subsequent process steps such as modification.

[0035] The present invention provides the above embodiments only to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments describe the present invention in detail, relevant technical personnel in the field should understand that the present invention can be modified or equivalently replaced, but any modification and partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for purifying a semaglutide precursor, characterized in that: The method first uses isoelectric precipitation to treat a crude precursor sample solution obtained after enzyme cleavage during the synthesis process, resuspends the precipitate, and then purifies it by hydrophobic chromatography to obtain a purified semaglutide precursor.

2. A method for purifying a semaglutide precursor according to claim 1, characterized in that: It includes the following detailed steps: Step S1, taking a crude sample solution of semaglutide precursor obtained after enzyme cleavage, adjusting the pH value of the crude sample solution to 4.8-5.0, allowing the solution to stand for precipitation, discarding the supernatant and collecting the precipitate, adding water to the precipitate to resuspend it, and adjusting the pH value of the resuspended solution to 7.0-9.0 to redissolve the precipitate to obtain a reconstituted solution; Step S2, using a chromatography column to separate and purify the complex solution obtained in step S1, the filler of the chromatography column is Generik MC60-HIC Butyl, and the chromatography treatment conditions are: Equilibration: Equilibrate the column with 2CV equilibration buffer; Sampling: Take the reconstituted solution and load it. The loading volume should not exceed 10g / L. Washing: Wash with 2.5CV equilibration buffer; Washing: Use 4CV washing buffer to clean impurities; Elution: isocratic elution with 2.5CV pure water, collecting the elution liquid corresponding to the main peak to obtain the purified semaglutide precursor.

3. A method for purifying a semaglutide precursor according to claim 2, characterized in that: In the step S1, the pH value of the crude sample solution is adjusted to 4.9, and the pH value of the resuspension solution is adjusted to 8.

0. The static precipitation time is greater than 20 minutes.

4. A method for purifying a semaglutide precursor according to claim 3, characterized in that: In step S1, the pH value is adjusted by using an inorganic acid or an inorganic base, wherein the inorganic acid includes one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and sodium dihydrogen phosphate, and the inorganic base includes one or more of sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonia water, calcium hydroxide, and sodium phosphate.

5. A method for purifying a semaglutide precursor according to claim 2, characterized in that: The equilibrium buffer is 450mM NaCl+45%-55% water with a pH value of 7.8-8.

8.

6. A method for purifying a semaglutide precursor according to claim 5, characterized in that: The equilibration buffer is 450 mM NaCl + 50% water with a pH of 8.

3.

7. A method for purifying a semaglutide precursor according to claim 2, characterized in that: The washing buffer is 450mM NaCl+80%-90% water with a pH value of 7.8-8.

8.

8. A method for purifying a semaglutide precursor according to claim 7, characterized in that: The washing buffer is 450 mM NaCl + 85% water with a pH value of 8.

3.

9. A method for purifying a semaglutide precursor according to claim 2, characterized in that: The method further comprises step S3, regenerating the chromatography column filler: regenerating with 2CV 0.5M NaOH and washing with 2CV pure water.

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