A method for purifying and oxidizing disulfide bond-containing polypeptides

A disulfide bond and oxidant technology, applied in the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of low purity, many impurities in solid phase oxidation, time-consuming and labor-intensive, etc., to improve yield and purity, and operate Simple, solve the effect of yield

Active Publication Date: 2020-11-20
HYBIO PHARMA WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are mainly the following methods for the disulfide bond oxidation of polypeptides. One is to oxidize the polypeptide during solid-phase or liquid-phase synthesis. Phase synthesis of linear crude peptides, after cleavage, dissolve the linear crude peptides, carry out liquid phase oxidation, and then perform purification such as: CN 103304655A, CN 104710509 A; Improve the purity of oxidation products or reaction efficiency, but each disulfide bond oxidation method has its advantages and disadvantages, solid-phase oxidation is more suitable for some short peptides or some polypeptides that only need to form a pair of disulfide bonds; liquid-phase oxidation is time-consuming Laborious, but the purity after oxidation is relatively high, which provides convenience for subsequent purification
In order to improve the safety of drugs, the higher the purity of the existing peptide drugs, the better. Most of them require a purity of more than 99%, and the control of single impurities is required. The single impurities are required to be less than 0.15%, and there are many solid-phase oxidation impurities, which is not conducive to quality. Control; if liquid-phase oxidation is used for purification, although the liquid-phase oxidation is complete and the purity is high, the oxidation treatment process is cumbersome and time-consuming
[0005] The invention provides a new liquid-phase disulfide bond oxidation method, which is different from the traditional oxidation method, and can make up for the shortcomings of low solid-phase oxidation yield and purity, and the time-consuming and labor-intensive shortcomings of the traditional liquid-phase oxidation method. It is easy to operate and greatly improves the oxidation purity. and yield, and easy to scale up production

Method used

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  • A method for purifying and oxidizing disulfide bond-containing polypeptides
  • A method for purifying and oxidizing disulfide bond-containing polypeptides
  • A method for purifying and oxidizing disulfide bond-containing polypeptides

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: Octreotide crude peptide purification

[0037] Dissolve and filter 2.0 g of octreotide linear crude peptide, and collect the filtrate for subsequent use.

[0038] Chromatographic column: octadecyl bonded silica gel chromatographic column, the column specification is 5cm×25cm.

[0039] Preparation Process:

[0040] Adjust the pH of 100 mmol / L potassium dihydrogen phosphate solution to 8.5 with ammonia water as mobile phase A, add 10 g of hydrogen peroxide to phase A, and mix well; use chromatographically pure methanol as phase B. With a flow rate of 60-80ml / min, the detection wave is 230nm. Phase B elution gradient: Gradient elution is performed on the sample with a gradient change of B%: 15%-35%, 50-70min. Purification is complete, while oxidation is complete.

[0041] The oxidized and purified polypeptide obtained in the above step is formed into a stable salt, and octreotide meeting the standard with a purity greater than 99.0% can be obtained after ...

Embodiment 2

[0043] Embodiment 2: Octreotide crude peptide purification

[0044] Dissolve and filter 15 g of octreotide crude peptide, and collect the filtrate for subsequent use.

[0045] 1 Chromatographic column: octadecyl bonded silica gel chromatographic column, the column specification is 10cm×25cm.

[0046] Adjust the pH of 100 mmol / L sodium dihydrogen phosphate solution to 8.0 with ammonia water as mobile phase A, and add 150 g of hydrogen peroxide; chromatographically pure methanol as phase B. Flow rate: 200ml / min. Detection wavelength: 230nm. Phase B elution gradient: Gradient elution is performed on the sample with a gradient change of B%: 8%-20%, 50-70min. Purification is complete, while oxidation is complete.

[0047] The oxidized and purified polypeptide obtained in the above step is formed into a stable salt, and octreotide meeting the standard with a purity greater than 99.0% can be obtained after freeze-drying.

[0048] After lyophilization, 6.1 g of white powdery soli...

Embodiment 3

[0049] Embodiment 3: Octreotide crude peptide purification

[0050] Dissolve and filter 25 g of octreotide crude peptide, and collect the filtrate for subsequent use.

[0051] Chromatographic column: octadecyl bonded silica gel chromatographic column, the column specification is 15cm×25cm.

[0052] Adjust the pH of 100mmol / L sodium dihydrogen phosphate solution to 7.5 with ammonia water, and add 150g hydrogen peroxide as phase A; chromatographically pure methanol as phase B. Flow rate: 200ml / min. Detection wavelength: 230nm. Phase B elution gradient: Gradient elution is performed on the sample with a gradient change of B%: 8%-20%, 50-70min. Purification is complete, while oxidation is complete.

[0053] The oxidized and purified polypeptide obtained in the above step is formed into a stable salt, and octreotide meeting the standard with a purity greater than 99.0% can be obtained after freeze-drying.

[0054] After lyophilization, 8.9 g of white powdery solid refined pept...

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Abstract

The present invention provides a method for purifying and oxidizing polypeptide containing disulfide bond simultaneously. Specifically, the crude peptide on the chromatographic column is rinsed with mobile phase A and phase B, wherein the phase A contains an oxidizing agent for oxidizing disulfide bond, therefore, the disulfide bond is oxidized during the purification, and after the purification, the oxidized polypeptide is obtained.

Description

technical field [0001] The invention relates to a purification method of polypeptide medicine. Background technique [0002] The disulfide bonds formed between one or more pairs of cysteine ​​residues play an important role in maintaining the spatial conformation of polypeptides and certain biological activities. Many existing drugs require the presence of disulfide bonds to be active, such as: octreotide, nesiritide, eptifibatide, atosiban and terlipressin, etc., so the oxidation method of disulfide bonds It has always been the focus of research in the peptide industry. [0003] At present, the conventional method for oxidizing polypeptide disulfide bonds is to first oxidize with air, hydrogen peroxide, iodine and other oxidants, and then use reversed-phase liquid chromatography to purify to obtain the final product. Although this method can complete the oxidation in 15min-30min, the oxidation speed is relatively fast, but after the oxidation reaction is stopped, purifica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/20C07K7/06C07K14/435C07K14/575C07K7/16C07K7/08
CPCC07K1/20C07K7/06C07K7/08C07K7/16C07K14/43509C07K14/575C07K14/435
Inventor 尹传龙宓鹏程陶安进袁建成
Owner HYBIO PHARMA WUHAN CO LTD
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