The reaction of salmon calcitonin (sCT) with various linear PEG and PolyPEGs was investigated under a variety of reaction conditions. The optimal experimental conditions for the production of high yield monoconjugated sCT were determined using RP-HPLC, SEC-HPLC, IE-FPLC and MALDI-TOF MS analysis. Semi-preparative IE-FPLC was used to purify high yield mono conjugated sCT-5 kDa linear PEG, sCT -6.5 kDa PolyPEG, sCT-20 kDa PolyPEG and sCT-40 kDa PolyPEG coupled to the N-terminal primary amine. The products were analysed using RP-HPLC, SEC-HPLC, SDS-PAGE and the site of attachment was determined by Tryptic mapping combined with LCMS, RP-HPLC and MALDI-TOF MS. Biological activity was measured invitro, sCT derivates were incubated with T47D cells and an ELISA assay was used to measure the amount of cAMP released. The purified conjugates retained a high degree of biological activity compared to the unmodified peptide. Incubation of sCT and sCTconjugates with proteolytic enzymes showed that for sCT-5 kDa linear PEG and 6.5kDa PolyPEG a high degree (60-70 % of the activity at time zero) of biological activity remained after 30 minutes compared to 0% activity for native sCT
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