Fig. 8.

Purification of mono- and diPEGylated amylin with mPEG-SC and mPEG-SPA in organic solvent. Amylin (5 mg/mL) was reacted in DMSO with ten times mPEG-SC (a, c, e) or mPEG-SPA (b, d, f) molar excess for 4 h at 25°C. The quenched reactions were purified by C18 RP-HPLC, which was followed by MALDI-ToF-MS analysis. a, b Chromatograms from the C18 RP-HPLC purifications. The peaks were collected and freeze-dried for further use. Free indicates the retention time region for unmodified amylin (the chromatogram of free amylin alone is not shown), peak I indicates the monoPEGylated amylin, peak II indicates the diPEGylated amylin, and peak III indicates the pooled remaining fractions containing other PEGylation products. Tail is the fraction collected between peaks “I” and “II,” and R is the quenched reaction before RP-HPLC purification. Inset, SDS-PAGE characterization of the RP-HPLC fractions. c, d MALDI-ToF-MS analysis of monoPEGylated amylin (peak I) e, f MALDI-ToF-MS analysis of diPEGylated amylin (peak II). The details are described in the “MATERIAL AND METHODS” section