High-yield production of biologically active mono-PEGylated salmon calcitonin by site-specific PEGylation

The purpose of this study was to develop and optimize a unique one-pot, two-step site-specific PEGylation method suitable for the high-yield production of mono-PEGylated (Lys¹⁸) salmon calcitonin (Lys¹⁸-PEG-sCT), which was previously demonstrated to have superior pharmaceutical properties to other conjugates. For the site-specific PEGylation, this study used the sCT derivative (FMOC^1,11-sCT), which was FMOC protected at Cys¹- and Lys¹¹-amines among three PEGylation sites including Lys¹⁸-amine. This PEGylation process was achieved by the consecutive one-pot, two-step reaction: (i) the PEG conjugation to FMOC^1,11-sCT; and (ii) the subsequent deprotection of FMOC group from the PEGylated FMOC^1,11-sCT. The optimized reaction resulted in the high production yield of Lys¹⁸-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys¹⁸-PEG-sCT conjugate showed improved biological stability without the loss in the in vitro and in vivo biological activity by PEGylation. Consequently, this site-specific PEGylation using an FMOC protection/deprotection strategy showed great usefulness in the production of the most promising Lys¹⁸-PEG-sCT conjugate with a high yield.