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Chromatographic purification method of fatty acid mono-acylation insulin

A chromatographic purification and insulin technology, which is applied in the field of chromatographic purification, can solve the problems of easy damage to the chemical properties of reversed-phase silica gel, affect the separation effect of fillers, troublesome maintenance, etc., and achieve good separation and purification effects, good cost advantages, and simple steps.

Active Publication Date: 2013-09-04
ZHUHAI UNITED LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alkaline washing can only be used to clean, but the alkaline solution is easy to destroy the chemical properties of the reversed-phase silica gel and affect the separation effect of the packing
At the same time, if RP-HPLC or high-pressure semi-preparative liquid-phase instruments are used as separation and purification instruments, although they are accurate and efficient, they are costly and troublesome to maintain, especially the cost is at least 50% higher than that of medium and low-pressure chromatography systems

Method used

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  • Chromatographic purification method of fatty acid mono-acylation insulin
  • Chromatographic purification method of fatty acid mono-acylation insulin
  • Chromatographic purification method of fatty acid mono-acylation insulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] (1) Preparation of samples to be purified:

[0056] Referring to Example 7 disclosed in patent WO98 / 02460, 0.50 g of Des B30 human proinsulin was dissolved in a mixed solution of 7.0 ml of dimethylsulfoxide (DMSO) and 3.5 ml of water, and 0.4 ml of triethylamine was added thereto Adjust the pH to 10.2-10.5 to obtain a mixed solution. Then dissolve 0.025g of N-succinimidyl myristic acid in 1.8ml of N-methylpyrrolidone solution, and add it dropwise to the aforementioned mixed solution, and finally place the homogeneously mixed solution at 0°C and stir for 2 hours to react , you can get insulin derivatives containing acylation modification, namely (N εB29 -tetradecanoyl) Lys B29 des (B30) reaction solution of human insulin. Then it was acidified and diluted 10 times with an aqueous solution containing acetic acid (the concentration of acetic acid is 10% by volume) to obtain the sample to be purified. The purity of the sample to be purified is as Figure 16 shown.

[...

Embodiment 2

[0067] (1) Preparation of the sample to be purified: same as step (1) in Example 1.

[0068] (2) Purification using QuickSep medium pressure chromatography system:

[0069] A chromatographic column with a diameter of 11 mm and a length of 250 mm is filled with 10 ml of nano-micro Uni PS30-300 filler (particle size 30 μm, pore size 300 angstroms), and the bed height is 110 mm.

[0070] The mobile phase preparation ratio is the same as in Example 1, and the pH of the mobile phase A is adjusted to be 3.0. Adjust the amount of loaded sample so that the loading of the target protein in the column is 4.0 g / L. Adjust the operating pressure of the chromatography system to be 2 bar, 5 bar and 8 bar respectively. The elution method is the same as that in Example 1. After the peak is detected by a 280nm UV detector, it is collected in sections, each section is 10ml, and then the components with a purity greater than 98.0% are mixed, then the purity and concentration are detected, and t...

Embodiment 3

[0078] (1) Preparation of the sample to be purified: same as step (1) in Example 1.

[0079] (2) Purification using QuickSep medium pressure chromatography system:

[0080] A chromatographic column with a diameter of 140 mm and a length of 500 mm is filled with 2.5 L of GE source30 filler (particle size 30 μm), and the bed length is 165 mm.

[0081] The chromatography buffer solution is: phase A, 0.15M sodium acetate buffer solution, pH 3.0; phase B, acetonitrile solution containing TFA (trifluoroacetic acid), the concentration of TFA is 0.1% by volume.

[0082] The column is first equilibrated with 10% B phase, that is, A phase and B phase are mixed at a volume ratio of 9:1. After equilibration, the sample is loaded so that the target protein load of the filler is 4.0g / L, and 40% B is eluted at an equal degree. The speed is 5.7L / h, the working pressure is 8bar, and the 280nm UV detector monitors the peak and collects it in sections, each section has a volume of 1.0L.

[008...

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Abstract

The invention discloses a chromatographic purification method of fatty acid mono-acylation insulin. The chromatographic purification method comprises the steps of separating the fatty acid mono-acylation insulin by employing a medium- and low-pressure chromatographic system, and using an organic polymer reversion phase chromatographic material as a stationary phase, and taking at least one organic solvent mixable with water and at least one buffering agent substance as an elution moving phase with the pH (Potential of Hydrogen) value of 2-4. The chromatographic purification method has simple steps, is easy to amplify, and low in economic cost, and can increase the purity of a sample from original 50-60% to 98% by one step.

Description

technical field [0001] The invention relates to a chromatographic purification method, in particular to a chromatographic purification method of fatty acid monoacylated insulin. Background technique [0002] Aminoacylation is a common protein modification method, and the general method of acylation is commonly found in "Methods of Enzyme" edited by C.H.W. Hirs and published by Elsevier, 25:494-499 (1972). [0003] At present, insulin detemir produced by Novo Nordisk, Denmark is a common insulin derivative obtained by using this principle to modify the ε-aminoacylation of Lys residue at position B29. In the aminoacylation reaction of insulin, active esters, acyl halides or acid anhydrides are generally used in the industry to react with proinsulin or analogs to prepare monoacylated insulin, but since proinsulin or analogs contain The free α-amino group of amino acid and the free ε-amino group on the side chain of other amino acids (such as the ε-amino group of Lys), these th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K1/20
Inventor 黄亮曹春来肖拥军曹永恒
Owner ZHUHAI UNITED LAB
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