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Sequence analysis method and identification method of insulin or analogue thereof

A technology for sequence analysis and insulin, applied in the field of sequence analysis method and identification of insulin or its analogs, can solve the problems of artificial modification of enzymatic hydrolysis reaction, long enzymatic hydrolysis reaction time, occurrence of interference peaks, etc., and achieves rapid reduction reaction and identification. Fast, accurate results

Inactive Publication Date: 2020-11-17
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1. Existing techniques require the use of expensive sequencing-grade restriction endoproteinases
[0008] 2. The enzymatic hydrolysis reaction time of the prior art is long, and it needs to be compared with the reference substance, and the identification cycle is long
[0009] 3. The existing technology enzymatic hydrolysis reaction has the risk of introducing artificial modification, resulting in interference peaks and affecting the judgment of results

Method used

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  • Sequence analysis method and identification method of insulin or analogue thereof
  • Sequence analysis method and identification method of insulin or analogue thereof
  • Sequence analysis method and identification method of insulin or analogue thereof

Examples

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Embodiment 1

[0069] The sequence analysis of embodiment 1 insulin aspart

[0070] Insulin aspart injection was diluted to 1 mg / mL with 10 mmol / L hydrochloric acid solution, 100 μL was taken into a centrifuge tube, and final concentrations of 6 mol / L guanidine hydrochloride and 50 mmol / L TCEP were added, and after incubation at 45°C for 40 min, the aspart The disulfide bond of insulin is reduced and opened, and the two peptide chains are separated. The reduced product was analyzed by ultra-high performance liquid chromatography-high resolution tandem mass spectrometry, and the injection volume was 2 μL. The instrumental analysis conditions are as described above. The preferred post-mass fragmentation mode of the insulin aspart A chain is HCD, and the preferred post-collision energy is 20; the preferred post-mass fragmentation mode of the insulin aspart B chain is HCD, and the preferred post-collision energy is 25.

[0071] The detection results of liquid chromatography-mass spectrometry a...

Embodiment 2

[0087] Example 2 Sequence Analysis of Insulin Degludec

[0088] Dilute insulin degludec injection with 10mmol / L hydrochloric acid solution to 1mg / mL, take 100μL into a centrifuge tube, add 6mol / L guanidine hydrochloride and 50mmol / L TCEP at final concentrations respectively, incubate at 45°C for 40min, and make degludec The disulfide bond of insulin is reduced and opened, and the two peptide chains are separated. The reduced product was analyzed by ultra-high performance liquid chromatography-high resolution tandem mass spectrometry, and the injection volume was 2 μL. The instrumental analysis conditions are as described above. The preferred post-mass fragmentation mode of the insulin degludec A chain is HCD, and the preferred post-collision energy is 20; the preferred post-mass fragmentation mode of the insulin degludec B chain is HCD, and the preferred post-collision energy is 25.

[0089] The detection results of liquid chromatography-mass spectrometry are as follows: F...

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Abstract

The invention relates to the field of drug analysis, in particular to a sequence analysis method and an identification method of insulin or analogues thereof. An appropriate disulfide bond reduction method is adopted, an insulin double-chain structure is reduced into two independent peptide chains, and the most abundant mass spectrum fragment information is obtained through liquid chromatography separation, high-resolution tandem mass spectrometry analysis and appropriate secondary mass spectrum collision energy and is matched with a theoretical sequence, so that the 100% sequence coverage rate is obtained; and a powerful technical support is provided for quality control and counterfeiting work of insulin.

Description

technical field [0001] The invention relates to the field of drug analysis, in particular to a sequence analysis method and identification method for insulin or its analogues. Background technique [0002] In the past ten years, biotechnology drugs have obvious advantages in the treatment of some diseases, and a large number of star drugs have emerged. Different from traditional small molecule chemical drugs, biotechnology drugs generally have the characteristics of large molecular weight, complex structure, poor stability, and large batch-to-batch variation. Therefore, their quality control is more complicated than that of chemical drugs, requiring a series of analytical techniques covering different analytical fields. , in order to effectively characterize its physical and chemical properties. The "Technical Guidelines for R&D and Evaluation of Biotechnological Drugs" (Trial) clearly requires that for protein and polypeptide drugs, the amino acid sequence of the candidate...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72
CPCG01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N2030/324
Inventor 王建伟孙跃权周奕含
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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