Reproducible preparation and effective separation of PEGylated recombinant human granulocyte colony-stimulating factor with novel "PEG-pellet" PEGylation mode and ion-exchange chromatography.
A novel preparation for polyethylene glycol (PEG) derivatives and chromatographic separation procedure of the PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were designed to evaluate the reproducibility and scalability at large laboratory-scale level. The new "PEG-pellet" PEGylation mode was successfully applied to control the pH fluctuation during the conjugation reaction, a general problem in traditional liquid-phase conjugation mode. Moreover, two consecutive ion-exchange chromatography steps were successfully used to separate and purify the PEGylated rhG-CSF. Cation-exchange chromatography was firstly applied to separate PEGylated rhG-CSF from intact rhG-CSF, followed by anion-exchange chromatography to obtain individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the excess free PEG. Furthermore, the molecular weight of individual PEGylated rhG-CSF was identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and SDS-PAGE, and cell proliferation activity in vitro was detected by MTT assay using NFS-60 cell.
Yun
Qiang
Q
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China.
Yang
Rui E
RE
Chen
Ting
T
Bi
Jingxiu
J
Ma
Guanghui
G
Su
Zhiguo
Z
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005591:Chemical Fractionation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015203:Reproducibility of Results
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols