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Hetergeneous product of bio-active protein and its preparing process

A biologically active, protein technology, applied in the covalent modification of amino acid residues of granulocyte colony-stimulating factor and water-soluble polymers, in the field of protein modification, which can solve the uncertainty of reaction sites, low clearance rate, kidney toxicity, etc. problems, to achieve the effect of being conducive to large-scale industrial production, reducing production costs, and simple production processes

Inactive Publication Date: 2007-06-13
赵剑
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high cost of this technology and the uncertainty of the reaction site, it brings great difficulties to the industrialization of PEG-modified proteins
Moreover, the biological activity of PEGylated proteins is reduced; the onset of action in the body is slow; and because of the low clearance rate in the body, repeated use is easy to cause toxicity to the kidneys

Method used

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  • Hetergeneous product of bio-active protein and its preparing process
  • Hetergeneous product of bio-active protein and its preparing process
  • Hetergeneous product of bio-active protein and its preparing process

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0024] 1. Preparation of recombinant human G-CSF

[0025] The amino acid sequence of human G-CSF is as follows:

[0026] Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys

[0027] Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu

[0028] Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu

[0029] Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly

[0030] Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu

[0031] Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala

[0032] Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu

[0033] Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly

[0034] Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu

[0035] Arg His Leu Ala Gln Pro

[0036] The inventor transformed the plasmid containing the sequence into Escher...

example 2

[0081] Dialyze the 1.5 mg / ml G-CSF solution with 100 mM (pH 8.0) sodium phosphate buffer overnight, and add 2-methoxymethoxymethyliminomethylpolyethylene glycol with an average molecular weight of 20,000 dissolved in the buffer Alcohol (molar ratio: polyethylene glycol:G-CSF=1:1), stirred slowly at 25°C for 5 hours.

[0082] The protein obtained by the above method was subjected to molecular sieve chromatography to remove unreacted polyethylene glycol. The column was Pharmacia SepHcryl S-200 HR, 300ml. Equilibrate with 20 mm sodium acetate (pH 4.0) buffer. The loaded protein was eluted at a flow rate of 6ml / min for 200 minutes, the outflow of the protein was monitored at 280nm, and the target protein peak was combined. The product was assayed by HPLC PEG-GCSF:G-CSF=15:85. The results of in vitro bioactivity assays are shown in Table 4.

example 3

[0084] Dialyze the 1mg / ml G-CSF solution with 100mM (pH8.0) sodium phosphate buffer overnight, and add 2-methoxymethyliminomethylpolyethylene glycol with an average molecular weight of 20000 dissolved in the buffer Alcohol (molar ratio: polyethylene glycol:G-CSF=6:1), stirred slowly at 4°C for 40 hours.

[0085] The protein obtained by the above method was subjected to molecular sieve chromatography to remove unreacted polyethylene glycol. The column was Pharmacia SepHcryl S-200 HR, 300ml. Equilibrate with 20 mm sodium acetate (pH 4.0) buffer. The loaded protein was eluted at a flow rate of 6ml / min for 200 minutes, the outflow of the protein was monitored at 280nm, and the target protein peak was combined. The product was determined by HPLC PEG-GCSF:G-CSF=85:15. The results of in vitro bioactivity assays are shown in Table 6.

[0086]The in vitro biological activity comparison of PEG-GCSF and G-CSF mixed preparations of different proportions of table 6

[0087] ...

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Abstract

A heterogeneous product of bioactiv-protein contains the polyglycol chemically modified G-CSF (15-85%) and G-CSF or other likes (15-85%), and the molecular wt. of polyglycol is 4000-50000 dalton. Its preparing process features the coupling reaction of G-CSF with polyglycol molecules in alkaline environment. Its advantages include simple process, just using one-step reaction, low cost, quickly taking its effect, high durability and stable aqueous solution of it.

Description

technical field [0001] The invention relates to the field of protein modification, in particular to the field of covalent modification of amino acid residues of granulocyte colony-stimulating factor (G-CSF) and water-soluble polymers. Background technique [0002] Granulocyte colony-stimulating factor (G-CSF) is a cytokine that promotes the growth of blood cells. It can promote the growth and hematopoiesis of granulocytes, and induce their differentiation, proliferation and maturation. It has obvious promoting effect on the recovery of neutropenia. As a whitening medicine, G-CSF has been widely used clinically at home and abroad. However, due to its high clearance in the body, patients must inject once a day to maintain effective blood levels. Modification of protein by PEG can solve this disadvantage of genetically engineered drugs (Mumtaz S, BachhawatBK, Indian J Biochem BiopHys 1991 Oct-Dec; 28(5-6): 346-51). Research on this aspect has also been published in national ...

Claims

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Application Information

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IPC IPC(8): C07K14/475A61K38/19A61P7/00A61P35/02
Inventor 赵剑金蓓文陈虎
Owner 赵剑