Deciphering Polymer Interactions in Bioconjugates with Different Architectures by Structural Analysis via Time‐resolved Limited Proteolysis Mass Spectrometry

Therapeutic proteins are commonly conjugated with polymers to modulate their pharmacokinetics but often lack a description of the polymer‐protein interaction. We deployed limited proteolysis mass spectrometry (LiP‐MS) to reveal the interaction of polyethylene glycol (PEG) and PEG alternative polymers with interferon‐α2a (IFN). Target conjugates were digested with the specific protease trypsin and a “heavy” 15N‐IFN wild type (IFN‐WT) for time‐resolved quantification of the cleavage dynamics. Interactions between IFN‐α2a and its high‐affinity receptor were detailed by LiP‐MS. Then, 10 kDa polymers of PEG, linear polyglycerol (LPG), and poly(2‐oxazoline) (POX) with two different cyclooctyne linkers (BCN/DBCO) were used for site‐specific bioconjugation to azide functionalized IFN‐α2a. Tryptic events at each cleavage site and in different structural environments (loops/helices) were compared. PEG and LPG were similar, and POX showed a reduced interaction profile with the IFN‐α2a surface. All‐atom molecular dynamics simulations of IFN‐DBCO‐polymer conjugates revealed distinct and transient (below 50 ns) protein‐interaction profiles for PEG, LPG, and POX.  Cleavage dynamics of IFN‐polymer conjugates from the BCN handle were homogeneous, pointing to a more conserved IFN structure than DBCO‐polymer conjugates. In summary, time‐resolved LiP‐MS for quantification of cleavage events enhances the structural understanding of transient IFN‐polymer interactions, which may be extended to other bioconjugate types.