| Granulocyte colony-stimulating factor (G-CSF) is a kind of lineage-specific growth factor that primarily influences the production and differentiation of neutrophils, and its use in medicine sometimes results in shortened periods of neutropenia in patients receiving intensive chemotherapy. Like many therapeutic protein drugs, because of enzymolysis and renal clearance, rhG-CSF shows short serum half-life, which necessitates multiple injections to maintain its blood drug level. PEGylation, the covalent modification of therapeutic proteins with PEG, can prolong the plasma serum half-life of proteins by increasing their hydrodynamic volume and hence decreasing their renal clearance. So the preparation of PEG modified rhG-CSF is contrituded in this article to prolong the short half-life of rhG-CSF without lowering its biologic activity.rhG-CSF forms as inclusion body during expressing in E.coli. The typical strategy used to recover an active protein from inclusion bodies usually involves isolation, washing, solubilization and finally refolding. The refolding process should be considered to be the key part to promote recovery of the bioactive protein. To improve the efficiency of refolding, we optimized the pH, time, temperature and the formula of refolding solution, including the ratio of urea, accurate concentration of arginine, oxidized/reduced glutathione and glycerol.rhG-CSF has many shortcomings that readily be hydrolyzed by enzymatic and eliminated by kidney, which result in short half-life in vivo and repeated daily injections. We adopted the method of PEG modification to expand the hydrodynamic volume of rhG-CSF and maintain its biological activity at the same time.After modification, the physic-chemical property of PEG modified rhG-CSF obviously changed comparing with original molecule. So temperature, pH and the selection of excipients are chosed to judge the stability of PEG-rhG-CSF, associating to forced degradation test, best storage condition of PEG-rhG-CSF is determined. |
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