Chromatographic removal combined with heat,acid and chaotropic inactivation of four model viruses

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.