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Special-shaped dual functional coupling agent-assisted PEG (polyethylene glycol) modifying method for protein

A bifunctional linker and modification method technology, applied in the field of protein chemistry, can solve the problems of unfavorable production control stability and high yield of PEG multi-modification, and achieve the effect of separation and purification and high yield of single modification

Inactive Publication Date: 2013-10-30
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

When random modification of amino groups is used, the multi-modification yield of PEG is too high, which is not conducive to production control and batch-to-batch stability

Method used

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  • Special-shaped dual functional coupling agent-assisted PEG (polyethylene glycol) modifying method for protein
  • Special-shaped dual functional coupling agent-assisted PEG (polyethylene glycol) modifying method for protein
  • Special-shaped dual functional coupling agent-assisted PEG (polyethylene glycol) modifying method for protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1SATA assists the PEG modification of RNaseA

[0030] experimental method

[0031] RNaseA was dissolved in 20 mM phosphate buffer solution (pH 7.4). SATA was first dissolved in N,N-dimethylformamide, and then a certain amount of 20mM phosphate buffer (pH 7.4) was added. According to the molar ratio of RNase A and SATA being 1:2 and 1:4, a certain amount of SATA solution was added to the RNaseA solution respectively, shaken and mixed, and left to stand at 4° C. for 3 hours to react.

[0032] Hydroxylamine was dissolved in 20 mM phosphate buffer (pH 7.4), and a certain amount of hydroxylamine solution was added to the mixture of RNaseA and SATA according to the molar ratio of RNaseA and hydroxylamine at a ratio of 1:20. After shaking, let stand at 4°C for 2 hours to react.

[0033] The reaction solution was transferred to an ultrafiltration tube with a molecular weight cut-off of 5kDa, and the centrifugation speed was set to 5000g to remove unreacted SATA, h...

Embodiment 2

[0038] The separation, purification and identification of embodiment 2PEG single modification product

[0039] experimental method

[0040] By means of dialysis, the above sample solution was replaced into 50 mM acetic acid-sodium acetate buffer solution (pH5.0, solution A). Using SP Sepharose High Performance cation exchange chromatography, the unreacted PEG in the sample was eluted with liquid A after loading the sample, and then 100% pH 5.0 was used, and 0.5M sodium chloride was added to the acetic acid-sodium acetate buffer solution ( Solution B) Elution of protein components. The eluted protein fractions were collected.

[0041] The PEG single-modified product of RNase A was separated and purified with a preparative chromatographic column Superdex 200 (2.6cm×70cm). The buffer system used was PBS buffer (pH 7.4), and the product was collected and concentrated.

[0042] The purified samples were identified by reducing SDS-PAGE. Using PolyA-PolyU sodium salt as the subs...

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Abstract

The invention relates to a method for assisting polyethylene glycol (PEG) to modify a protein by utilizing a special-shaped dual functional coupling agent N-succinimidyl-S-acetylthioacetate (SATA for short). The method comprises the following main modifying steps of: reacting the SATA with amino of the protein, introducing protected sulfydryl onto a protein molecule; reacting the SATA-modified protein with hydroxylamine, and removing a protection agent to expose the sulfydryl; carrying out covalent linkage on the protein-introduced sulfydryl and polyethylene glycol maleimide, so as to generate a PEG modifying product for the protein. The method has the advantages that, with respect to other polyethylene glycol modifying reactions, the yield of the mono-modified product is relatively high and can maximally reach 72%, no di-modified or multi-modified product is generated, the production efficiency of simulative amplifying in protein drugs is beneficially improved, and meanwhile, the production cost is lowered.

Description

technical field [0001] The invention belongs to the field of protein chemistry, and relates to a method for the PEG modification of proteins by a heterobifunctional linker N-succinimidyl-S-acetylthioacetate (SATA). The invention can effectively inhibit the PEG modification of proteins. The production of modified products and multi-modified products improves the yield of single modified products. Background technique [0002] Protein (polypeptide) drugs play an important role in clinical diagnosis, treatment or prevention of various diseases as vaccines. However, peptide and protein drugs have the disadvantages of low solubility, poor stability and short half-life. In addition, some peptide and protein drugs have antigenicity and immunogenicity, which limits the development and clinical application of peptide and protein drugs. [0003] Protein chemical modification technology refers to the covalent reaction of active groups on proteins (polypeptides) with modifiers such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96
Inventor 胡涛王俊苏志国马光辉刘慎翔
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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