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Enzymatic hydrolysis peptide map analysis method for polyethylene glycol modified protein medicine

A technology of polyethylene glycol and analysis method, which is applied in the field of peptide map analysis, and can solve problems such as the absence of PEG-modified peptides and incomplete enzymatic hydrolysis.

Inactive Publication Date: 2018-07-10
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to solve the technical problems of incomplete enzymatic hydrolysis and no PEG-modified peptides when the existing peptide map analysis method is applied to polyethylene glycol-modified proteins

Method used

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  • Enzymatic hydrolysis peptide map analysis method for polyethylene glycol modified protein medicine
  • Enzymatic hydrolysis peptide map analysis method for polyethylene glycol modified protein medicine
  • Enzymatic hydrolysis peptide map analysis method for polyethylene glycol modified protein medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 The peptide map analysis method of the present invention is applied to ASP and PEG-ASP

[0079] 1. Detection method

[0080] (1), enzymolysis:

[0081] The sample to be tested is desalted and replaced with 1% NH 4 HCO 3 In buffer (PH7.8);

[0082] Dilute the sample to 1.0 mg / mL, take 100 μL, and heat at 100 °C for 5 min;

[0083] 2ug trypsin (1mg / ml, add 2μl) per 100ug heated sample solution, digest at 37℃ for 16-24h;

[0084] Termination of the reaction: 10 μl of 50% acetic acid solution was added to the reaction system at 1:10 (v / v) to terminate the reaction;

[0085] PEG-ASP enzymatic hydrolysis blank and ASP enzymatic hydrolysis blank (that is, unenzymatically hydrolyzed PEG-ASP and ASP samples were prepared in the same way, with 2 μL of 1% NH 4 HCO 3 buffer (pH ~7.8) in place of 2 μL of enzyme).

[0086] (2), RP-HPLC detection

[0087] Chromatographic column: XBridge Protein BEH C4 column (4.6×250mm,

[0088] Mobile phase: A: 0.1% TFA + water / ...

Embodiment 2

[0100] Example 2 The peptide map analysis method of the present invention is applied to polyethylene glycol modified arginine deiminase (PEG-ADI)

[0101]1. Enzymatic hydrolysis:

[0102] To 500 μL of PEG-ADI sample, 20 μL of 0.5mM DTT and 20 μL of 2M urea were added, and the reaction was denatured at room temperature for 30 min. Desalination replacement with 1% NH 4 HCO 3 In the solution, 100 μL was taken and heated at 100 °C for 5 min. After cooling, 2 μg trypsin was added, and the enzyme was hydrolyzed at 37°C for 16 to 24 hours. The reaction was terminated by adding 10 μL of 50% acetic acid to the system at 1:10 (V / V). The processed samples were centrifuged for later use.

[0103] 2. RP-HPLC detection:

[0104] Chromatographic conditions:

[0105] Mobile phase: A: 0.1% TFA + water / acetonitrile (95:5, v / v); B: 0.1% TFA + water / acetonitrile (35:65, v / v)

[0106] Chromatographic column: XBridge Protein BEH C4 column (4.6×250mm,

[0107] The gradients are shown in T...

Embodiment 3

[0115] Example 3 The peptide map analysis method of the present invention is applied to polyethylene glycol-modified KLK1 (PEG-KLK1)

[0116] 1. Enzymatic hydrolysis

[0117] 1) Concentrate the enzyme solution containing the PEG-KLK1 sample by centrifugal ultrafiltration, centrifuge at 4000 rpm for 30 min, take 0.3 M Tris-HCl (pH 7.8, containing 1 M TCEP HCl and 10 mM EDTA) solution to resuspend the protein, dissolve, and resuspend the protein. The sample concentration was brought to about 1.0 mg / mL.

[0118] 2) Add 1:25 (v / v) μL of 0.5M iodoacetic acid to the reaction system, and react at room temperature for 30 minutes in the dark.

[0119] 3) Add 1:50 (v / v) μL of 0.5M DTT to the reaction system and mix well.

[0120] 4) The above mixture was taken and passed through a G25Desalting column for desalting, and replaced in Tris-HCl (pH 7.8) buffer solution containing 2M urea.

[0121] 5) Take 100 μL of the desalted mixture, add 2 μg trypsin at a ratio of 1:50 (v / v) (dissolve ...

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Abstract

The invention relates to a peptide map analysis method for polyethylene glycol modified protein. According to the method, firstly, a high-order structure of the protein needs to be destroyed to fullyexpose restriction enzyme cutting site, and then proteolysis is carried out; secondly, by selecting an appropriate chromatographic packing aperture, the peptide map analysis method with ideal analysisperformance for PEG-modified proteins is obtained. Through effective use of the method, a PEG-modified peptide segment in a peptide map can be effectively detected, and the technical problem that modified protein and unmodified protein cannot be distinguished by conventional methods is solved.

Description

technical field [0001] The invention relates to a peptide map analysis method suitable for PEGylated proteins, in particular to a peptide map analysis method suitable for PEGylated medicinal protease, more specifically a PEGylated asparaginase, Peptide Mapping Methods for Kininogenase or Arginine Deiminase. Background technique [0002] Medicinal proteins and polypeptides generally have shortcomings such as poor biological stability, short in vivo half-life and immunogenicity. Genetic engineering and chemical modification are usually used to modify them to overcome the above shortcomings. Polyethylene glycol (PEG) is a linear, freely coilable, uncharged polymer in solution, which is nontoxic, weakly antigenic and has good biocompatibility. Using it to covalently modify proteins can increase the circulating half-life of proteins in vivo and reduce their antigenicity, increase the solubility of proteins and change the biological distribution of proteins in the human body. Si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/89
CPCG01N30/89
Inventor 马永颜莎王俊
Owner ZONHON BIOPHARMA INST
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