Abstract
We studied the kinetics of PEGylation by observing the conjugation reaction between tresyl monomethoxy polyethylene glycol (TM-PEG) and free lysine. We employed a fluorescence assay and graphic methods to determine the reaction orders with respective to TM-PEG and lysine. According to the experimental results, the reaction orders of PEG and lysine are 1.69 and 1.09, respectively. We also determined the activation energy of PEGylation using Arrhenius’ theory and discussed the temperature sensitivity of PEGylation.The reaction kinetics was applied to optimize that PEGylation reaction to modify adenovirus. By raising reactant concentrations and with the addition of 5 ﹪sucrose in the reaction mixture, the loss of adenoviruses titer during PEGylation process could be reduced. Although adenovirus titer was reduced during PEGylation, PEGylated adenovirus exhibits better stability under harsh storage conditions. Our results showed that there was approximately 0.5 to 1 log loss of the original titer in PEGylated adenovirus per hour at 37℃ whereas there was at least 1 log per hour reduction in viral titer when non-PEGylated adenovius was incubated at 37℃. Similarly, PEGylated adenovirus is more stable than non-PEGylated adenovirus at 4 ℃; the rate of titer loss is 1.5 log/day and 1.0 log/day for non-PEGylated and PEGylated adenoviruses, respectively.