Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Abstract

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.