Figure 2.

N-glycans improve serum stability with minimal impact on GDF15 activity. (a) Summary of Fc-GDF15 functional activity and glycan occupancy, from highest intrinsic activity to lowest. Functional activity was measured by Phospho-ERK HTRF (Homogeneous Time Resolved Fluorescence) assay and reported as EC₅₀ and % intrinsic activity (maximum response, Ymax) normalized to wildtype Fc-GDF15 (Fc-WT). Data are reported as mean ± SD. N = 4. EC₅₀ values of “n/a” indicate that EC₅₀ could not be determined due to low activity. Glycan occupancy at engineered NxT sites was measured using capillary gel electrophoresis. (b) Representative data from p-ERK assay summarized in (a). N = 4. The Y-axis values represent the HTRF signal ratio (665/615 nm × 10⁴). (c) In vitro serum stability of wildtype Fc-GDF15 control (Fc-WT), Mutant 2, Mutant 3, and Mutant 7 following 4 days of incubation in mouse serum or a PBS control at 37 °C. Gross clipping was detected by Western blot using an anti-human Fc antibody. Clipped chain highlighted in red. Full length blots are presented in Supplementary Fig. 6.