两步连续离子交换制备色谱分离纯化聚乙二醇化重组人粒细胞集落刺激因子

Separation and Purification of PEGylated rhG-CSF by Two-step Ion-exchange Chromatography

建立了两步连续离子交换制备色谱分离、纯化聚乙二醇与重组人粒细胞集落刺激因子 (Recombinanthumangranulocytestimulatingfactor,rhG_CSF)偶联物的方法。首先用阳离子交换色谱将偶联蛋白质和非偶联蛋白质分开 ,然后使用阴离子交换色谱去除过量的游离聚乙二醇杂质 ,并分离纯化偶联蛋白异构体分别得到单聚乙二醇化、双聚乙二醇化和三聚乙二醇化的rhG_CSF。它们经十二烷基磺酸钠_聚丙烯酰胺凝胶电泳 (SDS_PAGE)分析均为单带。采用基质辅助激光解吸离子化_飞行时间质谱(MALDI_TOF)分析三种偶联蛋白质的分子量 ,分别为 2 3 8kD、2 8 6kD、33 8kD。用噻唑蓝 (MTT)比色法 ,以粒细胞集落刺激因子的依赖细胞株NFS_6 0为靶细胞 ,测定重组人粒细胞集落刺激因子及其与聚乙二醇的偶联物的体外细胞生物学活性 ,单聚乙二醇化、双聚乙二醇化和三聚乙二醇化的rhG_CSF体外活性保留率分别为 92 %、75 %、4 3%。

In order to separate and purify the PEGylated recombina nt human granulocyte stimulating factor (rhG-CSF) at large laboratory-scale leve l, a two-step ion-exchange chromatographic separation procedure was designed. Ca tion-exchange chromatography was applied first to separate PEGylated rhG-CSF fro m un-reacted rhG-CSF, followed by anion-exchange chromatography to dissolve indi vidual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the free PEG. The molecular weight of individual PEGylated rhG-CSF was determined b y MALDI-TOF and SDS-PAGE. MALDI-TOF mass spectrometry revealed that the molecul ar weights of mono-, di- and tri-PEGylated rhG-CSF are 23.8kD, 28.6kD and 33. 8kD, respectively. Cell proliferation activity was detected by MTT assay using N FS-60 cell. The in vitro residual bioactivity of mono-, di- and tri-PEGylated rhG-CSF were 90%, 75% and 43% respectively, comparing with the un -conjugated rhG-CSF. These results indicated that the un- conjugated rhG-CSF and excess free PEG can be removed completely and the three c onjugate species can be purified into homogeneity by the two consecutive ion-exc hange chromatographic steps. The purification procedure is easy to scale-up, hig h in performance and recovery.