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 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.