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

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cydooctyne linkers (BCN/DBCO) for bioconjugation of interferon-alpha 2a (IFN-alpha 2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-alpha 2a was azide fimctionalized via amber codon expansion and bioorthogonally conjugated to all cydooctyne linked polymers. Polymer conjugation did not impact IFN-alpha 2a's secondary structure and only marginally reduced IFN-alpha 2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-alpha 2a 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-alpha 2a 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.

Automated summary

Conjugating biologics with different polymers and cydooctyne linkers showed that PEtOx and LPG bioconjugates had similar biological outcomes to PEG, indicating potential as alternatives for bioconjugation.