Preparation and characterization of compstatin Cp40 and long-acting derivatives thereof. (A) Schematic representation of the PEGylation strategy: PEG-Cp40 was prepared by adding a reactive two-arm branched PEG moiety of 40 kDa to the unprotected amino terminus of Cp40. In the case of Ac-Cp40-K-PEG, the N terminus was acetylated, a C-terminal lysine residue was added to the peptide during synthesis, and the PEG reagent was reacted with the lysine side chain. The dipeptide fragment used for the mass spectrometric quantification of PEG-Cp40 in plasma is indicated in red. (B) Evaluation of the complement inhibitory activity of Cp40 and its PEGylated derivatives using a complement activation ELISA. The clinically developed analog 4(1MeW)¹⁴  is shown as a control (for more information on individual compstatin analogs, see also supplemental Figure 1). The panel shows a representative plot out of 3 separate experiments.