Pharmacokinetic evaluation of PEG-Cp40 in NHPs. (A) Dose scheme for PEG-Cp40 administration: a single dose of 200 mg PEG-Cp40 was injected intravenously into 2 cynomolgus monkeys at time 0 (green arrow) and blood samples were drawn at various time points (red arrows). Given the size difference between Cp40 (1.7 kDa) and PEG-Cp40 (∼40 kDa), the selected dose corresponds to ∼2 mg of active peptide per kg. (B) Monitoring of PEG-Cp40 plasma concentrations as determined by ultra performance liquid chromatography-high definition mass spectrometry (after fragmentation using subtilisin-A and SPE). Baseline levels of plasma C3 (measured by ELISA in T0 sample) are depicted as dotted lines for each animal. Note that the 1 and 2 hour postinjection blood draws were not included in this analysis. (C) Change of plasma C3 levels during the treatment with PEG-Cp40; western blot analysis after 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (reducing conditions) was performed with plasma samples collected at different time points and a polyclonal C3 antibody was used for detection (top). Although the antibody recognized both chains of C3, the reactivity with the α-chain was generally higher. Plasma levels of an unrelated protein (transferrin) were used as an internal control on the same membrane after stripping and reprobing with a transferrin antibody (bottom). Panel shows a representative blot from at least 2 independent analyses of samples from 2 animals each.