Preparation and characterization of PEGylated adducts of recombinant human tumor necrosis factor-alpha from Escherichia coli.
This study was conducted to get an insight into monomethoxypolyethylene glycol (MPEG) modified recombinant human TNF-alpha (rhTNF-alpha) derived from gene cloning and expression. The purification and modification processes were integrated together to make the scale-up production of PEG-modified rhTNF-alpha practical. Capillary electrophoresis was demonstrated to be a highly efficient tool in the biochemical characterization of the PEGylated products compared to slab electrophoresis. The cytotoxicity of MPEG-modified rhTNF-alpha was studied in vitro towards L929 cell line and found to decrease gradually along with the increase in the reaction ratio between the activated MPEG and the native rhTNF-alpha. The MPEG-modified rhTNF-alpha was more resistant to proteinase degradation in vitro than the native. When MPEG chains were released partially from the MPEG-modified rhTNF-alpha by alkaline pretreatment, the cytotoxicity of the MPEG-modified rhTNF-alpha was enhanced, which was in contrast to the native. These results would be helpful to explain the disagreement between the high bioavailability of MPEG-modified TNF-alpha in vivo and its decreased cytotoxicity in vitro.
Li
Weijun
W
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, People's Republic of China. weijunli@yahoo.com
Wang
Yunshan
Y
Zhu
Xiaonan
X
Li
Ming
M
Su
Zhiguo
Z
D000818:Animals; D001709:Biotechnology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D014357:Trypsin; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity
C092033:PEG-modified tumor necrosis factor-alpha; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D014357:Trypsin