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Identification method for O-glycosylation site of insulin and analogues thereof

A technology of glycosylation site and identification method, applied in the field of identification of insulin and its analogs O-glycosylation site, can solve the problems of high cost, high price, low purchase amount and the like, and achieves strong practicability, High accuracy and simple operation effect

Active Publication Date: 2020-02-28
DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although ETD technology can also be used for the identification of glycosylation sites, this technology is not very stable, and its application is not as wide as that of CID. For peptides with a large number of amino acids, it is necessary to obtain relatively complete c / z ions. Currently, Only thermo's orbitrap fusion and waters' Synapt G2-S Q-TOF can be equipped with ETD modules, and ECD can only be used in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), but the prices of these two mass spectrometers They are quite expensive, the price is more than 5 million, and the purchase volume is relatively small. Generally, pharmaceutical companies or outsourcing institutions do not have this type of mass spectrometry
However, only by conventional enzymatic digestion methods, such as the peptide map of V8 enzyme digestion, LC-MS analysis can only roughly determine which peptide is glycosylated, and perform Q-TOF-MS / In MS analysis, the specific site of glycosylation cannot be confirmed because the glycosylated peptide contains multiple potential sites and the glycosidic bond is easy to break
[0003] Based on the traditional mass spectrometry fragmentation methods such as collision fragmentation (CID) that will preferentially fragment sugar chains and produce fewer peptide backbones, the fragmentation methods of ECD and ETD are more expensive, and conventional enzyme digestion techniques alone cannot To confirm the specific glycosylation site, therefore, it is urgent to develop a method with high accuracy, simple operation, low cost and new type of O-glycosylation site identification in insulin and its analog products

Method used

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  • Identification method for O-glycosylation site of insulin and analogues thereof
  • Identification method for O-glycosylation site of insulin and analogues thereof
  • Identification method for O-glycosylation site of insulin and analogues thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Elastase Enzyme Reducing Peptide Map Enzyme Cutting Treatment Time Investigation

[0086] Elastase is a serine-like protease that preferentially cleaves the C-terminus of alanine, valine, serine, glycine, leucine, or isoleucine. Peptide EQCCTS (A3-A9) can theoretically be obtained by cleavage of recombinant human insulin with elastase, but the theoretical peptide EQCCTS has not been obtained through preliminary experimental results, which may be due to valine, leucine, isoleucine Dipeptide derivatives of acid and alanine are potent competitive inhibitors of Elastase.

[0087] It has been reported in the literature that Elastase has multiple active centers and can cut many sites, but it only preferentially cuts the C-terminus of alanine, valine, serine, glycine, leucine or isoleucine and Elastase has the ability to degrade elastin activity, as well as the activity of hydrolyzing protein.

[0088] In order to obtain key fragment ion peaks for subsequent mass s...

Embodiment 2

[0096] Example 2 Q-TOF-MS / MS CID Fragmentation Energy Optimization

[0097] MS / MS analysis was performed on the key glycosylated peptides in the glycosylated impurity Elastase digestion and reduction peptide map, and the peptide with m / z688.2518 (CTSIC glycosylation) was used as the parent ion, and the CID of the peptide was Fragmentation energy has been optimized. During the experiment, it was found that when performing MS / MS analysis, when the energy is too low, the desired target fragments cannot be obtained, and when the energy is too high, the sugar is easily broken during the process of CID fragmentation, and the target cannot be obtained fragment ions.

[0098] Chromatographic conditions: Column information: Welch C18, 4.6mm×250mm, 5μm (No.: 20121108); Injection volume: 20μl; Wavelength: 214nm; Column temperature: 40°C; Flow rate: 1.0ml / min; Flow into the mass spectrometer; 0~4min: to waste; 4~50min: to MS; post-run time: 12min; gradient elution: elution time 0~50min,...

Embodiment 3

[0103] Example 3 Confirmation of glycosylation sites of recombinant insulin glargine glycosylation impurities

[0104] The glycosylation impurities (6225Da) in the crude drug of recombinant insulin glargine (molecular weight 6063Da) were confirmed by Elastase enzymatic hydrolysis.

[0105] By comparing the Elastase enzymatic profile of insulin glargine glycosylation impurities and insulin glargine standard after reduction, in the TIC profile of glycosylation impurities, it is found that the abundance of glycosylated peptides at m / z 688.2532 is higher, which may be CTSIC or CCTSI glycosylated peptides, but the corresponding glycosylated peptide peaks were not found in the spectrum of the glargine standard. In order to determine the glycosylation site, the glycosylated peptide was subjected to Target MS / MS analysis showed that m / z688.2532 was identified by Target MS / MS as a CTSIC glycosylated peptide and the glycosylation site was on threonine at position A8.

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Abstract

The invention relates to the field of biochemistry, in particular to an identification method for the O-glycosylation site of insulin and analogues thereof. The method comprises the following steps: performing elastase digestion on insulin and analogues thereof, analyzing a peptide map through LC-MS, and preliminarily determining a corresponding glycosylated peptide fragment; and carrying out Q-TOF-MS / MS analysis on the key glycosylation peptide fragment, optimizing CID mass spectrum fragmentation energy of the key peptide fragment to obtain key fragment ions with sugar, and analyzing the O-glycosylation site more accurately, simply and conveniently. According to the method, glycosylation site analysis and confirmation of complex peptide fragments can be realized under a conventional CID fragmentation technology, the method is simple to operate, high in accuracy, high in practicability, economical and suitable for all mass spectrometers with the CID fragmentation technology, and a newthought is provided for post-translational modification analysis of glycosylation and application in proteomics.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for identifying O-glycosylation sites of insulin and its analogues. Background technique [0002] Recombinant human insulin and its analogs are irreplaceable products for the treatment of diabetes, but in the process of large-scale production of recombinant products in biopharmaceuticals, post-translational modifications of proteins usually result. Insulin analogues generally use yeast expression system, and a relatively common O-glycosylation modification will be formed during the expression process. During process development, the proportion of O-glycosylation modification should be reduced as much as possible, and the structure of O-glycosylation impurities should be identified. O-glycosylation modifications generally occur on threonine (T) or serine (S). The amino acid sequences of insulin products contain more T and S, and the identification of O-glycosylation sites is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/68
CPCG01N33/74G01N33/6848G01N2333/62
Inventor 龚庆伟冯艳蒋燕封启媛徐军谢灿高相雷李晓平陈小锋李文佳
Owner DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD