聚乙二醇对PEG化重组人促红细胞生成素热稳定性和生物学活性的影响

为了研究聚乙二醇对PEG化rhEPO热稳定性和生物学活性的影响,用酶学方法切除rhEPO和PEGrhEPO分子中的N连接糖基,研究酶切产物在37℃时287nm和350nm吸光度随时间的变化;用MTT比色法,网织红细胞法和HCT法比较体内和体外生物学活性的改变。结果显示聚乙二醇修饰能显著减少无N连接糖基的rhEPO分子间的聚集,降低rhEPO体外生物学活性,但增高体内生物学活性;无N连接糖基的PEG化rhEPO具有与rhEPO相当的体内生物学活性。因此聚乙二醇能提高rhEPO热稳定性;聚乙二醇可替代糖基,维持rhEPO的体内生物学活性。用PEG修饰原核细胞表达的rhEPO是开发rhEPO制品的新思路。

The effect of polyethylene glycol on the thermostability and biological activity of PEGylated rhEPO

To study the effect of PEG on thermostability and biological activity of PEGylated rhEPO, the thermostability of the enzymatic N-deglycosylation products was studied by monitoring their absorbance on 350nm or 287nm at 37℃. The biological activity was evaluated by using MTT colorimetry, measuring the increase of haematocrit and reticulocyte counts. PEG modification can significantly reduce the aggregation tendency among rhEPO molecules which have been digested with PNGase F to remove N-linked oligosaccharides, increase biological activity in vivo but reduce of the preparation in vitro severely. The biological activity in vitro of PEG-rhEPO that lost all N- linked carbohydrate was comparable with that of the rhEPO. PEG can increase the thermostability of rhEPO and substitute of oligosaccharides to sustain its biological activity in vivo. Therefore, it is feasible to develop a novel rhEPO which has therapeutic value by means of prokaryotic expression and PEGylation.