[Separation and purification of PEGylated rhG-CSF by two-step ion-exchange chromatography].
In order to separate and purify the PEGylated recombinant human granulocyte stimulating factor (rhG-CSF) at large laboratory-scale level, a two-step ion-exchange chromatographic separation procedure was designed. Cation-exchange chromatography was applied first to separate PEGylated rhG-CSF from un-reacted rhG-CSF, followed by anion-exchange chromatography to dissolve individual 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 by MALDI-TOF and SDS-PAGE. MALDI-TOF mass spectrometry revealed that the molecular weights of mono-, di- and tri-PEGylated rhG-CSF are 23.8 kD, 28.6kD and 33.8kD, respectively. Cell proliferation activity was detected by MTT assay using NFS-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 conjugate species can be purified into homogeneity by the two consecutive ion-exchange chromatographic steps. The purification procedure is easy to scale-up, high in performance and recovery.
Chen
Ting
T
Civil & Environmental Engineering School, University of Science and Technology of Beijing, Beijing 100083, China.
Yun
Qiang
Q
Bi
Jing-Xiu
JX
Ma
Guang-Hui
GH
Su
Zhi-Guo
ZG
D002852:Chromatography, Ion Exchange / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols