CHARACTERIZATION AND STABILITY OF N-TERMINALLY PEGYLATED RHG-CSF

Purpose. The liquid stability of rhG-CSF was investigated after polyet hylene glycol (PEG) with an average molecular weight of 6000 daltons w as covalently attached to the N-terminal methionine residue. Methods. The conjugation methods chosen for modifying the N-terminal residue we re alkylation and acylation. The N-terminally PEGylated rhG-CSF conjug ates were purified by cation exchange chromatography. The physical cha racterization methods of SDS-PAGE, endoproteinase peptide mapping, cir cular dichroism and in-vivo bioassay were used to test for differences between the PEG-rhG-CSF molecules. Results. Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temper atures, of these conjugated molecules indicated that the primary pathw ay of degradation was aggregation. Conjugation through alkylation offe red the distinct advantage of decreasing, by approximately 5 times, th e amount of aggregation present as compared to acylation. Conclusions. We suggest, that the increased stability observed for the molecules u tilizing the alkylation conjugation method may be due to the preservat ion of charge on the alpha amino group of rhG-CSF.