Study On The Preparation, Purification, Characterization And Bioactivity Of PEGylated Recombinant Human Growth Hormone

OBJECTIVE To investigate the optimal reaction condition of modifying rhGH by mPEG-Propionaldehyde(mPEG-ALD) with relative molecular weights of 20kD and the method for the purification of mono-PEG-rhGH. And to study the characterization and biological activity of the mono-PEG-rhGH. METHODS rhGH was modified with mPEG-ALD under various conditions, the ratio of mono-PEG-rhGH was determined by SDS-PAGE and thin layer scanning. The reaction mixture was purified by ion-exchange and size exclusion chromatography. The MALDI-TOF-MS , Size Exclusion HPLC, CIEF, Circular Dichroism and Fluorescence Spectrometry were used to study on the characterization of the mono-PEG-rhGH, including its relative molecular weight, purity, isoelectric point, and its secondary structure. The location of the conjugates was determined by peptide mapping. The receptor binding activities were evaluated with their abilities to inhibit the binding of ~125I-hGH to growth hormone receptor on the membrane of pregnant rabbit liver cells. And body weight gain tests of hypophysectomized rats were carried out for potency determination. RESULTS pH 6.0,1:8 for the mass ratio of rhGH and PEG, 16 hours for reaction time was elected as the optimal reaction condition of modification of rhGH with PEG.The purity of mono-mPEG-rhGH purified by ion-exchange and size exclusion chromatography was higher than 98%. There is no apparent difference in the physical characterization of the mono-PEG-rhGH comparing with the rhGH. Its relative molecular weight was 43 KD,and its isoelectric points was 5.2. The PEGylation sites was the α -NH2 of theN-terminal amino. The receptor binding activity of the mono-PEG-rhGH decreased ,while injection of mono-PEG-rhGH every 6 days over a 12-day period was even more effective than rhGH given daily. CONCLUSION :The optimal reaction condition of modifying rhGH by mPEG-ALD and the separation methods of the reaction mixture was primarily determined. Studies of the characterization and biological activity support the use of PEGylation to extend the activity of rhGH that are normally cleared by filtration and provide promising long-acting alternatives to daily hGH injections.