Conjugation Strategy Strongly Impacts the Conformational Stability of a PEG-Protein Conjugate

Site-specific PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, the impact of these different conjugation strategies on the properties of PEG–protein conjugates is poorly understood. Here we show that the ability of PEG to enhance protein conformational stability depends strongly on the identity of the PEG–protein linker, with the most stabilizing linkers involving conjugation of PEG to planar polar groups near the peptide backbone. We also find that branched PEGs provide superior stabilization relative to their linear counterparts, suggesting additional applications for branched PEGs in protein stabilization.