Anna Mero, Oddone Schiavon, Mariano Schiavon, Francesco M. Veronese and Gianfranco Pasut
Department of Pharmaceutical Sciences, University of Padua, Via F. Marzolo 5, 35131 Padua, Italy firstname.lastname@example.org
PEGylation is widely studied to prolong protein half-life and to decrease their immunogenicity. To date, the requirements for the approval of new conjugates are stringent, and obtaining a single PEG-protein isomer or at least a wellcharacterized mixture of mono-PEGylated isomers is mandatory. Protein enzymatic PEGylation can achieve this goal, thanks to the high specificity of specific enzymes. It has been already demonstrated that microbial transglutaminase (mTGase) is an interesting enzyme for site-specific modification. TGase can link PEG-NH2 to only one or few protein Gln(s), a selectivity dictated by specific substrate requirements. However, even this approach losses its advantages in those cases having at least two Glns as TGase substrates, because a mixture of mono and bi-conjugates is generated. In this study, we investigated the role of cosolvents in the reaction mixture that, by modifying the secondary structure of the target protein, allows the obtainment of a specific monoconjugate. The method was studied on two model proteins, sCT and hGH. For sCT, the in vivo activity of the conjugate was also studied in rats.