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.

Polyglycerols as Multi-Functional Platforms: Synthesis and Biomedical Applications

“…This can be carried out either by applying different initiators or postmodification of the end group subsequent to the polymerization process [5,33,36,[83][84][85]. LPG has been successfully conjugated to proteins via different chemistries, including random conjugation to bovine serum albumin (BSA) [33], site-specific CuAAC to Exenatide, N-terminally to Interleukin-4 [86], and Anakinra [87] and to IFN-α-2a via SPAAC [88]. These studies clearly demonstrated a comparable size of PEGylated and LPGylated proteins with a similar half-life extension in vivo.…”

Polymer selection impacts the pharmaceutical profile of site specifically conjugated Interferon-α2a

“…Polymer synthesis of LPG and PEG was performed as described previously [49]. In brief, LPG was synthesized by monomer-activated anionic ring-opening polymerization.…”

“…Therefore, we recently synthesized bioconjugates through strain-promoted azide-alkyne cycloaddition (SPAAC), starting off a genetically engineered, azido-IFN-α2a mutant and functionalized PEG, and LPG polymers of about 10 kDa, respectively [49]. All products were generally comparable across the two polymer chemistries reported here, including their preferential interaction sites with the protein-surface.…”

“…More recently, biologics have been introduced facilitating sitespecific enzymatic catalysis, an approach which uses a biologic's intrinsic substrate site or, if not available, a genetically introduced substrate site through which functionalized polymers are enzymatically conjugated [31][32][33][34][35]. Alternatively, genetical introduction of unnatural amino acids, guarantees polymer decoration at that preselected site [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52]. As to the second, immunological challenges of PEGylated biologics, recent reports point to possible side-effects including hypersensitivity, [53,54] and immunological responses, [28,55] which are at least in part driving accelerated plasma clearance after repetitive dosing [56].…”

“…As to the second, immunological challenges of PEGylated biologics, recent reports point to possible side-effects including hypersensitivity, [53,54] and immunological responses, [28,55] which are at least in part driving accelerated plasma clearance after repetitive dosing [56]. Hence, expanding the polymer space beyond PEGylation is of much interest and linear polyglycerol (LPG) has been discussed in this context before [49,51,52].…”