A method for in situ enzymatic cleavage and purification of liraglutide fusion protein

Abstract

The invention discloses a method for in-situ enzyme digestion and purification of a liraglutide fusion protein. The method comprises the following steps: fixing a liraglutide fusion protein containing a His tag by a nickel affinity chromatography method, completing in-situ enzyme digestion of the liraglutide fusion protein on a nickel affinity chromatography column, and realizing separation of the fusion protein containing the His tag and a liraglutide main chain in one step; recovering enterokinase by GST affinity chromatography, and further improving the purity by reverse phase chromatography to obtain the liraglutide main chain. The product recovery rate is improved by 30.26%, the product purity is improved by 6.18%, the production cost is saved, the purification efficiency is improved, and economic value is created for enterprises.

Description

Technical Field

[0001] The invention relates to the field of drug preparation, and in particular to a method for in situ enzymatic cleavage and purification of a liraglutide fusion protein. Background Art

[0002] Liraglutide was originally developed by Novo Nordisk and was launched in Denmark in 2009. Liraglutide injection was approved by EMA and FDA in June 2009 and January 2010, respectively, for the treatment of type 2 diabetes. Liraglutide is a GLP-1 analog. Lys at position 34 on the human GLP-1 (7-37) chain is replaced by Arg, and glutamine modified with hexadecanoic acid is connected to Lys at position 26. After modification with adipose linkage, the affinity with albumin is increased, thereby reducing the hydrolysis rate and renal clearance rate by DPP-IV, extending the biological half-life to 11-15 hours, and only one subcutaneous injection is required per day, which greatly improves patient compliance.

[0003] The purification and enzymatic cleavage process of liraglutide requires membrane filtration, nickel affinity chromatography, reverse phase purification and other treatments to obtain high-purity liraglutide fusion protein, which is then enzymatically cleaved and purified to obtain the liraglutide backbone. The process is very complicated, resulting in a low recovery rate of the liraglutide backbone and high purification costs. Therefore, it is particularly important to develop a low-cost, easy-to-use enzymatic cleavage and purification technology. Summary of the invention

[0004] The object of the present invention is to provide a method for in-situ enzymatic cleavage and purification of a liraglutide fusion protein, by immobilizing the liraglutide fusion protein, completing the enzymatic cleavage on a nickel affinity chromatography column, and realizing the separation of the liraglutide main chain in one step, thereby reducing the purification steps, improving the purification efficiency and recovery rate, and reducing the product cost.

[0005] The technical solution of the present invention is as follows:

[0006] The His-tagged liraglutide fusion protein was obtained through preliminary filtration and purification. After fully loading the column (17.05 mg / mL) on a 5 ml nickel affinity chromatography column, impurities were washed with buffer. 20 ml of enterokinase solution was prepared and circulated into the nickel affinity chromatography column. In situ enzyme cleavage was performed at room temperature for 1 to 4 hours. After the enzyme cleavage was completed, the circulating liquid was recovered. The circulating liquid was passed through the NW Rose GST 4B chromatography column to adsorb enterokinase, and the liraglutide flow-through was collected. The flow-through was subjected to reverse phase purification, and the high-purity liraglutide backbone was collected and detected using HPLC.

[0007] The liraglutide fusion protein of the present invention comprises a HIS tag, an enterokinase sequence, an enterokinase cleavage site, liraglutide and other structures, wherein liraglutide accounts for 20.05% (m / m).

[0008] The buffer solution shown in the present invention includes 20mM Tris-HCl pH=6.5~7.5, 50mM NaCl, and 2mM CaCl2.

[0009] The enterokinase solution of the present invention is prepared by dissolving enterokinase in a buffer system, and the amount of enterokinase added is 17-340 U / mL.

[0010] The enterokinase shown in the present invention has a GST tag.

[0011] The circulating fluid of the present invention contains Tris-HCl, NaCl, CaCl2, liraglutide main chain and enterokinase.

[0012] The flow-through solution of the present invention contains Tris-HCl, NaCl, CaCl2 and a liraglutide main chain.

[0013] The detection using HPLC described in the present invention has the following advantages compared with the prior art:

[0014] By using fixed target products for in-situ enzymatic cleavage, a method for in-situ enzymatic cleavage and purification of liraglutide fusion protein was obtained. Its innovation lies in fixing the liraglutide fusion protein containing His tag by nickel affinity chromatography, completing in-situ enzymatic cleavage on the nickel affinity chromatography column, and separating the fusion protein containing His tag and the liraglutide main chain in one step, and then recovering enterokinase by GST affinity chromatography, and further improving the purity by reverse phase chromatography to obtain the liraglutide main chain, the product recovery rate increased by 30.26%, the product purity increased by 6.18%, saving production costs, improving purification efficiency, and creating economic value for enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the chromatogram of liraglutide fusion protein.

[0016] Figure 2 This is the main chain chromatogram of liraglutide obtained in Example 2.

[0017] Figure 3 The main chain chromatogram of liraglutide obtained in comparative example. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0019]

[0020] Example 1

[0021] The His-tagged liraglutide fusion protein was fully loaded onto a 5 ml nickel affinity chromatography column (17.05 mg / mL), and impurities were washed out using a buffer solution (20 mM Tris-HCl, pH=6.5, 50 mM NaCl, 2 mM CaCl2); 20 mL of enterokinase solution was prepared at an addition amount of 17 U / mL, and the enterokinase solution was circulated and injected into the nickel affinity chromatography column, and in-situ enzyme cleavage was performed at room temperature for 1 hour; after the enzyme cleavage was completed, the circulating liquid was recovered; the circulating liquid was passed through a NW Rose GST 4B chromatography column to adsorb enterokinase, and the liraglutide flow-through was collected. The flow-through was subjected to reverse phase purification, and a high-purity liraglutide backbone was collected. The obtained liraglutide backbone was tested by HPLC, and its purity was 94.13%, and the recovery rate was 47.50%, which was 16.01% higher than that of the comparative example.

[0022] Example 2

[0023] The His-tagged liraglutide fusion protein was fully loaded onto a 5 ml nickel affinity chromatography column (17.05 mg / mL), and impurities were washed out using a buffer solution (20 mM Tris-HCl pH=7.0, 50 mM NaCl, 2 mM CaCl2); 20 mL of enterokinase solution was prepared at an addition amount of 170 U / mL, and the enterokinase solution was circulated and injected into the nickel affinity chromatography column, and in-situ enzyme cleavage was performed at room temperature for 2 hours; after the enzyme cleavage was completed, the circulating liquid was recovered; the circulating liquid was passed through a NW Rose GST 4B chromatography column to adsorb enterokinase, and the liraglutide flow-through was collected. The flow-through was subjected to reverse phase purification, and a high-purity liraglutide backbone was collected. The obtained liraglutide backbone was tested by HPLC, and its purity was 95.61%, and the recovery rate was 61.75%, which was 30.26% higher than that of the comparative example.

[0024] Example 3

[0025] The His-tagged liraglutide fusion protein was fully loaded onto a 5 ml nickel affinity chromatography column (17.05 mg / mL), and impurities were washed out using a buffer solution (20 mM Tris-HCl pH = 7.0, 50 mM NaCl, 2 mM CaCl2); 20 mL of enterokinase solution was prepared at an addition amount of 340 U / mL, and the enterokinase solution was circulated and injected into the nickel affinity chromatography column, and in-situ enzyme cleavage was performed at room temperature for 2 hours; after the enzyme cleavage was completed, the circulating liquid was recovered; the circulating liquid was passed through a NW Rose GST 4B chromatography column to adsorb enterokinase, and the liraglutide flow-through was collected. The flow-through was subjected to reverse phase purification, and a high-purity liraglutide backbone was collected. The obtained liraglutide backbone was tested by HPLC, and its purity was 94.82%, and the recovery rate was 56.75%, which was 25.26% higher than that of the comparative example.

[0026] Example 4

[0027] The His-tagged liraglutide fusion protein was fully loaded onto a 5 ml nickel affinity chromatography column (17.05 mg / mL), and impurities were washed out using a buffer solution (20 mM Tris-HCl pH=7.0, 50 mM NaCl, 2 mM CaCl2); 20 mL of enterokinase solution was prepared at an addition amount of 170 U / mL, and the enterokinase solution was circulated and injected into the nickel affinity chromatography column, and in-situ enzyme cleavage was performed at room temperature for 4 hours; after the enzyme cleavage was completed, the circulating liquid was recovered; the circulating liquid was passed through a NW Rose GST 4B chromatography column to adsorb enterokinase, and the liraglutide flow-through was collected. The flow-through was subjected to reverse phase purification, and a high-purity liraglutide backbone was collected. The obtained liraglutide backbone was tested by HPLC, and its purity was 95.22%, and the recovery rate was 61.54%, which was 30.05% higher than that of the comparative example.

[0028] Example 5

[0029] The His-tagged liraglutide fusion protein was fully loaded onto a 5 ml nickel affinity chromatography column (17.05 mg / mL), and impurities were washed out using a buffer solution (20 mM Tris-HCl pH = 7.5, 50 mM NaCl, 2 mM CaCl2); 20 mL of enterokinase solution was prepared at an addition amount of 170 U / mL, and the enterokinase solution was circulated and injected into the nickel affinity chromatography column, and in-situ enzyme cleavage was performed at room temperature for 2 hours; after the enzyme cleavage was completed, the circulating liquid was recovered; the circulating liquid was passed through a NW Rose GST 4B chromatography column to adsorb enterokinase, and the liraglutide flow-through was collected. The flow-through was subjected to reverse phase purification, and a high-purity liraglutide backbone was collected. The obtained liraglutide backbone was tested by HPLC, and its purity was 94.62%, and the recovery rate was 57.13%, which was 25.64% higher than that of the comparative example.

[0030] Comparative Example

[0031] The His-tagged liraglutide fusion protein was fully loaded onto a 5 ml nickel affinity chromatography column (17.05 mg / mL), and 400 mM imidazole was used for linear elution to collect the fusion protein, and the imidazole was removed by a 10 kd ultrafiltration membrane to obtain a liraglutide fusion protein solution, and 3400 U enterokinase was added to perform enzyme digestion for 2 h at room temperature; after the enzyme digestion, the enzyme digestion solution was loaded onto a chromatography column filled with NW Rose GST4B filler, enterokinase was adsorbed, and the liraglutide flow-through was collected. The flow-through was subjected to reverse phase purification to collect the high-purity liraglutide backbone. The obtained liraglutide backbone was tested by HPLC, and its purity was 89.43%, and the recovery rate was 31.49%.

[0032] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for in situ enzymatic cleavage and purification of liraglutide fusion protein, characterized in that: Nickel affinity chromatography column was used for enzyme digestion and purification. The specific steps are as follows: 17.05 mg / mL His-tagged liraglutide fusion protein is fully loaded onto a 5 ml nickel affinity chromatography column, and impurities are washed away with a buffer; wherein the buffer is 20 mM Tris-HCl pH=6.5-7.5, 50 mM NaCl, 2 mM CaCl2; Prepare 20 mL of enterokinase solution at an addition amount of 17-340 U / mL, inject the enterokinase solution into the nickel affinity chromatography column in a loop, and perform in situ enzyme digestion at room temperature for 1 to 4 hours; After the enzyme digestion is completed, the circulating liquid is recovered; the circulating liquid is passed through the NW Rose GST 4B chromatography column to adsorb enterokinase, and the liraglutide flow-through is collected. The flow-through is subjected to reverse phase purification to collect the high-purity liraglutide backbone; the buffer is 20mMTris-HCl pH=7.0, 50mM NaCl, 2mM CaCl2; The amount of enterokinase added is 100-300U / mL; In situ cleavage of liraglutide fusion protein using enterokinase in a nickel affinity column; The enzyme cutting time is 1.5-3h.

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