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Modification method of polyethyleneglycol of protein

A technology of PEGylation and polyethylene glycol, applied in the field of protein modification, can solve the problems of inapplicable functional proteins, limited application scope, inability to carry out PEG modification reaction, etc.

Inactive Publication Date: 2013-12-04
WENZHOU MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, through a large number of previous studies, it has been found that the existing ion exchange or affinity chromatography solid-phase modification is not suitable for all functional proteins. As far as the ion-exchange chromatography solid-phase modification technology is concerned, the possible reason is that the adsorption protein and The electrostatic interaction between the ionic and solid phases shields the PEG modification site, resulting in the inability of the PEG modification reaction; while affinity chromatography requires the target protein to be able to specifically bind to the chromatographic column. The binding force with the chromatographic column puts forward higher requirements, which greatly limits the application range

Method used

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  • Modification method of polyethyleneglycol of protein
  • Modification method of polyethyleneglycol of protein
  • Modification method of polyethyleneglycol of protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Solid-phase PEG modification of lysozyme based on hydrophobic chromatography column

[0022] Include the following steps:

[0023] (1) Pre-equilibrate the HiTrap Butyl FF column (column volume CV=1mL) with phosphate buffer PB (20mM, pH6.0, 2MNaCl) containing 2M NaCl.

[0024] (2) Use the above PB buffer (20mM, pH6.0, 2M NaCl) to prepare lysozyme solution with a concentration of 1mg / mL lysozyme solution, take 5mL and load it on the HiTrap Butyl FF chromatographic column at a flow rate of 1mL / min. After sample loading, wash with PB buffer (20mM, pH6.0, 2M NaCl) until no protein breaks through.

[0025] (3) To contain 30mM NaBH 3 Prepare 10mL of 2mg / mL polyethylene glycol-butyraldehyde (20kDa) solution in PB buffer (20mM, pH6.0, 2MNaCl) of CN, and use the mPEG-butyraldehyde solution as the mobile phase at a flow rate of 1mL / min Pass through the HiTrap Butyl FF chromatographic column, keep the column temperature at room temperature, and wrap the HiTrap Butyl FF...

Embodiment 2

[0030] Example 2 Solid-phase PEG modification of lysozyme based on hydrophobic chromatography column

[0031] Include the following steps:

[0032] (1) Pre-equilibrate the HiTrap Phenyl FF column (column volume CV=1mL) with phosphate buffer PB (20mM, pH6.0, 2MNaCl) containing 2M NaCl.

[0033] (2) Use the above PB buffer (20mM, pH6.0, 2M NaCl) to prepare lysozyme solution with a concentration of 1mg / mL lysozyme solution, take 5mL and load it on the HiTrap Phenyl FF chromatographic column at a flow rate of 1mL / min. After sample loading, wash with PB buffer (20mM, pH6.0, 2M NaCl) until there is no breakthrough.

[0034] (3) To contain 30mM NaBH 3 Prepare 10mL of 2mg / mL polyethylene glycol-butyraldehyde (5kDa) solution in PB buffer (20mM, pH6.0, 2MNaCl) of CN, and use the mPEG-butyraldehyde solution as the mobile phase at a flow rate of 1mL / min Pass through the HiTrap Phenyl FF chromatographic column, keep the column temperature at room temperature, and wrap the HiTrap Phenyl FF...

Embodiment 3

[0039] Example 3 α-chymotrypsin solid-phase PEG modification based on hydrophobic chromatography column

[0040] Include the following steps:

[0041] (1) Pre-equilibrate the HiTrap Phenyl FF column (column volume CV=1mL) with phosphate buffer PB (20mM, pH6.0, 2MNaCl) containing 2M NaCl.

[0042] (2) Use the above PB buffer (20mM, pH6.0, 2M NaCl) to prepare α-chymotrypsin into a protein solution with a concentration of 1mg / mL, take 5mL and load it on a HiTrap Phenyl FF column at a flow rate of 1mL / min, After the sample loading is completed, wash with PB buffer (20mM, pH6.0, 2M NaCl) until there is no breakthrough.

[0043] (3) To contain 30mM NaBH 3 Prepare 10mL of 2mg / mL polyethylene glycol-propionaldehyde (20kDa) solution in CN's PB buffer (20mM, pH6.0, 2MNaCl), and use this solution as the mobile phase to flow through HiTrap Phenyl FF at a flow rate of 1mL / min For chromatographic column, keep the column temperature at room temperature, and wrap the HiTrap Phenyl FF chrom...

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Abstract

The invention relates to modification method of polyethyleneglycol of protein, which takes hydrophobic chromatography chromatographic column as solid phase reaction carrier. The method comprises the following steps: as for the protein with strong hydrophobicity, absorbing the protein on the hydrophobic chromatography chromatographic column under a high-salt state, taking a polyethyleneglycol modification agent as a mobile phase, and taking salt ion for gradient elution after the completion reaction to obtain polyethyleneglycol modified protein; as for the protein with hydrophobicity, absorbing the polyethyleneglycol modification agent in the hydrophobic chromatography chromatographic column under the high salt sate, taking protein solution as the mobile phase, and taking salt ion for gradient elution after the completion reaction to obtain polyethyleneglycol modified protein. The PEG modified protein prepared by the invention has high retention rate of activity and better simplicity, and the single modified rate reaches more than 40 percent, and the retention rate of activity reaches more than 80 percent. The method provides a new process for the realization of PEG modification of the protein.

Description

technical field [0001] The invention belongs to the field of protein modification, in particular, it relates to a protein pegylation modification method. Background technique [0002] With the development of genetic engineering technology, more and more attention has been paid to the clinical application of protein and polypeptide drugs. However, protein and polypeptide drugs generally have defects such as poor stability, short half-life in vivo, and immunogenicity, which seriously restrict their application and development. Polyethylene glycol (PEG) can improve the dissolution behavior of protein drugs, enhance stability, eliminate or reduce immunogenicity, prolong the half-life of in vivo circulation, and optimize pharmacokinetics, so it has important applications in biotechnology and biopharmaceutical development value and broad application prospects. At present, PEG has been applied to the modification of many protein drugs, such as PEG-modified asparaginase, interfero...

Claims

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Application Information

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IPC IPC(8): C07K1/113C07K1/04
Inventor 黄志锋牛建楼李校堃刘白玲宋林涛谢遥遥朱雁林施璐
Owner WENZHOU MEDICAL UNIV
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