Figure 3. FpF_infliximab preparations: assessment of stability, affinity & intracellular uptake.

(a) Representative Coomassie and silver-stained SDS-PAGE demonstrate the synthesis steps and protein size modifications involved in synthesis of FpF_infliximab from whole infliximab (lanes 1–7). FpF_infliximab stored at 4 °C for 480 days did not display dissociation from the PEG linker or aggregate formation (lane 8), (b) SPR binding analysis demonstrates that FpF_infliximab binds murine TNF-α using NTA chip, (c) Kinetic affinity (K_D) and rate constants (k_a, k_d) for infliximab and FpF_infliximab to recombinant human TNF-α. FpF has similar binding affinity to full IgG, but the dissociation rate constants are 5× slower for FpF_infliximab, (d) Whole cell lysates prepared from B6RPE-07 cells treated with infliximab or FpF_infliximab, for 4 & 24 hrs, were probed and the presence of the molecules detected using anti-human IgG Fab. As a negative control, cells were incubated with media alone; and as a positive control preparations of either molecule were applied to the lane.