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Purification method and application of liraglutide

A liraglutide and purification method technology, applied in the field of liraglutide purification, can solve the problems of high cost, high price, large sample volume, etc., achieve shortened service life, increase service life, and reduce economic costs Effect

Active Publication Date: 2020-06-12
THE UNITED BIO-TECH (HENGQIN) CO LTD +1
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent CN104592381A discloses a method for preparing a liraglutide intermediate peptide, which is a method for preparing a liraglutide intermediate peptide using fusion protein inclusion bodies, and discloses the use of Q-FF anion exchange for rough purification, Then use C8 reverse-phase packing to purify and obtain the method of liraglutide intermediate peptide. This method uses ion exchange chromatography for purification. After the inclusion body is denatured and dissolved, a large amount of refolding buffer must be added for refolding treatment, and the sample is loaded. Large volume, not convenient for industrial production; C8 is a reversed-phase silica gel filler, which is expensive and expensive compared to general chromatography materials

Method used

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  • Purification method and application of liraglutide
  • Purification method and application of liraglutide
  • Purification method and application of liraglutide

Examples

Experimental program
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preparation example Construction

[0105] Preparation of liraglutide mixture liquid:

[0106] Referring to Example 6 disclosed in Chinese patent CN107881187A, dissolve 2000 g of liraglutide fusion protein inclusion bodies in 15 L of guanidine hydrochloride solution with a concentration of 6 mol / L, add 0.8 L of DMSO and 150 ml of DIEA, mix well, and the pH is 11.37; Then add 50mg / ml N α - 1.1L of DMSO solution of hexadecanoyl-Glu(ONSu)-OH (side chain of liraglutide, CAS number: 294855-91-7), stirred and reacted for 3 hours; then added 17L of 20mmol / L Tris solution, and adding 9000IU lysyl-specific endonuclease, dilute hydrochloric acid or sodium hydroxide solution to adjust the pH value to 9.30, after 8 hours of reaction, samples were taken for HPLC detection, and a total of 18.85 g of liraglutide was obtained. HPLC detection spectrum such as figure 1 As shown, the purity of liraglutide was 30.14%.

Embodiment 1

[0108] pH value experiment of liraglutide mixture feed liquid: the pH value of liraglutide mixture feed liquid was adjusted to 2.0, 3.0, 4.0, 6.0 with hydrochloric acid, and the experimental phenomena are shown in Table 3:

[0109] table 3

[0110] pH2.0 pH3.0 pH4.0 pH5.0 pH6.0 precipitation massive precipitation massive precipitation massive precipitation clear liquid

[0111] The theoretical isoelectric point of liraglutide is around pH 5.0, and the liraglutide mixture feed solution prepared from the inclusion body fusion protein exists in a precipitated state at pH 2.0 to 5.0, and cannot be chromatographically purified; when the pH value Clear solution when reaching 6.0.

Embodiment 2

[0113] (1) Chromatographic purification

[0114] Chromatography packing material: Nanwei Uni NM100 (average particle size 100μm, pore size );

[0115] Purification system: QuickSep medium pressure chromatography system;

[0116] Chromatographic column: 100mm×500mm, packing volume 2L.

[0117] Take 17L of the prepared liraglutide mixture feed solution, add 4.25L of isopropanol, adjust the pH value to 7.0 with hydrochloric acid (6mol / L), and load it to a 5L equilibrium solvent (which contains 20mmol / L Tris, 20% v / v isopropanol, pH7.0) equilibrated chromatographic column, equilibrate the chromatographic column with 3L equilibrating solvent, use elution solvent (containing 20mmol / L Tris, 40% v / v isopropanol, pH7.0 ) for elution, the elution flow rate is 40cm / h, the pressure is 10bar, under the condition of ultraviolet 280nm, when the UV value rises, the components containing liraglutide are collected, and the collection volume is 2.5L. figure 2 is the chromatogram of the pur...

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Abstract

The invention discloses a purification method and application of liraglutide. The method comprises the following steps: separating a liraglutide-containing mixture by using a medium-low pressure reversed phase chromatography system to obtain liraglutide, wherein the stationary phase is an organic polymer reversed-phase chromatographic material, the mobile phase for elution contains at least one organic solvent miscible with water and at least one pH buffer substance, and the pH value is 6-9. According to the method, high-purity liraglutide can be obtained through medium-low pressure reversed-phase chromatography, an organic polymer reversed-phase filler which is convenient to use and low in price is adopted, and compared with other purification means, the method has the great cost advantage and is suitable for industrial preparation of liraglutide.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for purifying liraglutide and its application. Background technique [0002] Liraglutide [Arg 34 Lys 26 -(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37)] is a GLP-1 (glucagon-like peptide-1) analog, in the treatment Type 2 diabetes has excellent results. At present, the production of liraglutide by genetic engineering is mainly expressed in Escherichia coli, and the method generally uses Arg 34 -GLP-1(7-37) fragment is expressed by fusing the molecular chaperone and connecting peptide, and then the molecular chaperone and connecting peptide are excised with specific tool enzymes to obtain the intermediate peptide Arg 34 -GLP-1(7-37) was modified with fatty acid to obtain liraglutide. As disclosed in Chinese patent CN106434717A, Arg is obtained by adding HIS tag affinity chromatography and purifying 34 -Soluble fusion protein of GLP-1(7-37), then cut with enterokina...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/20
CPCC07K14/605Y02A50/30
Inventor 曹永恒陈亮航黄亮武振军曹春来周翠夏志成谢鑫邓慧兴
Owner THE UNITED BIO-TECH (HENGQIN) CO LTD
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