Site - specific PEGylation of rHuEPO and evaluation of its biological activity and stability

Despite the critical role of erythropoietin (EPO) as therapeutic agent in treatment of anemia, its consumption is limit ed due to several disadvantages including the product short half-life, immunogenicity and susceptibility to proteolytic degradation. To overcome these drawbacks efficient methods such as site-specific PEGylation have been developed among witch N-terminal PEGylation has found more interest due to its capability to preserve the protein native structure. In this study a site-specific PEGylation of rHuEPO is conducted and the biological activity and the stability of the modified protein are evaluated. rHuEPO was N-Terminally PEGylated with PEG-propionaldehyde through Schiff base reaction in acidic pH conditions. The product was then purified to yield in mono N-terminally PEGylated EPO. The resultant PEG-EPO and unmodified EPO was then compared to evaluate the consequence of the pegylation process. The results showed that despite the lower activity of PEG-EPO relative to the unmodified EPO, the earlier gained improved stability as a consequence of site-specific PEGylation. This observation provides further indication for the usefulness of PEGylation approach to enhance characteristics of erythropitein.