Separation and purification of PEGylated rhG-CSF by two-step ion-exchange chromatography.
- Author:
Ting CHEN
1
;
Qiang YUN
;
Jing-Xiu BI
;
Guang-Hui MA
;
Zhi-Guo SU
Author Information
1. Civil & Environmental Engineering School, University of Science and Technology of Beijing, Beijing 100083, China.
- Publication Type:Journal Article
- MeSH:
Chromatography, Ion Exchange;
methods;
Granulocyte Colony-Stimulating Factor;
biosynthesis;
chemistry;
isolation & purification;
Humans;
Polyethylene Glycols;
chemistry;
isolation & purification;
Recombinant Proteins
- From:
Chinese Journal of Biotechnology
2005;21(2):284-288
- CountryChina
- Language:Chinese
-
Abstract:
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.
