title: High-yield production of biologically active mono-PEGylated salmon calcitonin by site-specific PEGylation.
abstract: 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 (Lys(18)) salmon calcitonin (Lys(18)-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 Cys(1)- and Lys(11)-amines among three PEGylation sites including Lys(18)-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 Lys(18)-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys(18)-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 Lys(18)-PEG-sCT conjugate with a high yield.
authors.affiliation: Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea.
authors.forename: Yu Seok
authors.initials: YS
authors.lastname: Youn
authors.forename: Dong Hee
authors.initials: DH
authors.lastname: Na
authors.forename: Kang Choon
authors.initials: KC
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002116:Calcitonin / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*; D002118:Calcium / Q000097:blood; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D005079:Excipients; D005449:Fluorenes; D006207:Half-Life; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C496789:9-fluorenylmethoxycarbonyl; D005079:Excipients; D005449:Fluorenes; D011092:Polyethylene Glycols; C028815:salmon calcitonin; D002116:Calcitonin; D000242:Cyclic AMP; D002118:Calcium