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A kind of peptide map analysis method of nesiritide and its application

An analytical method, nesiritide technology, applied in analytical materials, material separation, instruments, etc., can solve problems such as early peaks, time-consuming, analytical deviations, etc.

Active Publication Date: 2022-07-05
SHANGHAI JINGFENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, in the peptide map analysis of nesiritide, trypsin lysis-reverse liquid chromatography is usually used to analyze it. After the analysis, the fragments decomposed by nesiritide need to be collected. However, this process is relatively time consuming
When assigning the peaks of the liquid chromatogram, it is often considered that some small molecular fragments (high polarity) appear behind relatively large molecules (small polarity). However, according to the retention mechanism of reversed-phase chromatography, the polarity Large fragments should be weakly retained on the reversed-phase chromatographic column, and the peaks will elute earlier, so there is a bias in the analysis of the prior art in terms of fragment assignment

Method used

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  • A kind of peptide map analysis method of nesiritide and its application
  • A kind of peptide map analysis method of nesiritide and its application
  • A kind of peptide map analysis method of nesiritide and its application

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Experimental program
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Effect test

Embodiment 1

[0056] This embodiment provides a method for analyzing peptide maps of nesiritide, which specifically includes the following steps:

[0057] (1) Reduction and alkylation of nesiritide samples

[0058] Dilute the sample with water to 1.5mg / mL, add it to a 1.5mL centrifuge tube, add 100μL 0.1mol / L ammonium bicarbonate buffer, then add 11.56μL 0.1mol / L DTT (dithiothreitol), mix well, and add Incubate at 56 °C for 1 h, and after incubation, place it in a 20 °C water bath to cool for 1 min.

[0059] Add 12.6 μL of 0.25 mol / L iodoacetamide solution to the reduced sample, mix well, and incubate at 20°C for 1 h in the dark.

[0060] After the reaction, use mass spectrometry to confirm that the reduction and alkylation are complete (e.g. figure 1 shown), four mass spectral peaks can be observed in the figure, with figure 2 The comparison of the mass spectra of the unreduced and alkylated nesiritide shows that the reduction and alkylation are complete, and the disulfide bond of nesi...

Embodiment 2

[0074] This embodiment provides a method for analyzing peptide maps of nesiritide, which specifically includes the following steps:

[0075] (1) Reduction and alkylation of nesiritide samples

[0076] Dilute the sample with water to 1.5mg / mL, add it to a 1.5mL centrifuge tube, add 100μL 0.4mol / L ammonium bicarbonate buffer, then add 11.56μL 0.4mol / L DTT (dithiothreitol), mix well, and add Incubate at 56 °C for 1 h, and after incubation, place it in a 25 °C water bath to cool for 1 min.

[0077] Add 12.6 μL of 0.5 mol / L iodoacetamide solution to the reduced sample, mix well, and incubate at 25°C for 1 h in the dark.

[0078] After the reaction, use MS detection to confirm that the reduction and alkylation are complete (such as Figure 4 shown), four mass spectral peaks can be observed in the figure, with figure 2 The comparison of the mass spectra of unreduced and alkylated in 2000 shows that the reduction and alkylation are complete, and the disulfide bond of nesiritide is c...

Embodiment 3

[0089] This embodiment provides a method for analyzing peptide maps of nesiritide, which specifically includes the following steps:

[0090] (1) Reduction and alkylation of nesiritide samples

[0091] Dilute the sample with water to 1.5mg / mL, add it to a 1.5mL centrifuge tube, add 100μL 0.2mol / L ammonium bicarbonate buffer, then add 11.56μL 0.2mol / L DTT, mix well, and incubate at 56°C for 1h, incubate After that, it was cooled in a 15°C water bath for 1 min.

[0092] Add 12.6 μL of 0.5 mol / L iodoacetamide solution to the reduced sample, mix well, and incubate at 15°C for 1 h in the dark.

[0093] After the reaction, use MS detection to confirm that the reduction and alkylation are complete (such as Figure 4 shown), four mass spectral peaks can be observed in the figure, with figure 2 The comparison of the mass spectra of unreduced and alkylated in 2000 shows that the reduction and alkylation are complete, and the disulfide bond of nesiritide is completely opened.

[0094...

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Abstract

The invention provides a peptide map analysis method of nesiritide and its application. The method includes the following steps: reducing and alkylating nesiritide, adding endopeptidase for enzymatic hydrolysis, and then using LC-UV-MS for detection, and analyzing the nesiritide according to the collected peptide map data. Polypeptide fragments and / or amino acid residues formed after tripeptidyl enzymatic hydrolysis are assigned. In the peptide map analysis method provided by the present invention, after the reduction and alkylation of nesiritide, endopeptidase is used to enzymatically decompose it into multiple polypeptide fragments and amino acid residues, and the nesiritide is processed by LC-UV-MS. The detection correctly corresponds the polypeptide fragments and amino acid residues to the peaks in the peptide map, so as to accurately determine whether the polypeptide chain of nesiritide has deletions, misconnections or duplications.

Description

technical field [0001] The invention belongs to the technical field of drug analysis, and in particular relates to a peptide map analysis of a recombinant polypeptide drug, in particular to a peptide map analysis method of nesiritide and its application. Background technique [0002] Heart failure is a clinical syndrome in which ventricular filling or ejection capacity is impaired due to structural abnormalities of the heart, and its main clinical manifestations are limited activity tolerance and fluid retention. The traditional treatment methods for heart failure are "cardiac, diuretic, and vasodilator", including diuretics, inotropes, beta-blockers, nitrates and other drugs, although they can relieve the clinical symptoms of heart failure patients, However, the efficacy is limited, and it cannot reverse myocardial fibrosis and ventricular remodeling caused by heart failure. Therefore, it is necessary to find new clinical drugs that can fundamentally treat heart failure. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8813
Inventor 李帅苏春所刘美卓芳芳
Owner SHANGHAI JINGFENG PHARMA
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