Structure-guided PEGylation of IL-1RA preserves fold and antagonist function

Abstract

High Resolution Image Download MS PowerPoint Slide Rational design of PEGylated protein therapeutics requires mechanistic understanding of how coupling chemistry and PEG size jointly determine product homogeneity, receptor engagement, and structural integrity─the properties that collectively govern whether a conjugate is a viable candidate for further development. Anakinra (recombinant interleukin-1 receptor antagonist, IL-1Ra) is an approved anti-inflammatory biologic whose short systemic residence necessitates daily subcutaneous injection and is associated with steep peak–trough exposure fluctuations that, in chronic high-dose regimens, have been linked to IL-1Ra-derived systemic amyloidosis. PEGylation offers a chemically established route to increase the apparent molecular size of IL-1Ra and thereby reduce renal filtration and smooth exposure, but the structural and functional consequences of conjugation have never been characterized at residue resolution for this target─a gap that limits informed design choices between available chemistries. Here, we address this gap by establishing a head-to-head characterization framework comparing site-specific thiol-maleimide conjugation and random amino-coupling on a uniformly 15 N-labeled recombinant IL-1Ra variant (M143V) that preserves all native conjugation sites and is potency-matched to Anakinra. Across a 5–20 kDa PEG ladder at a substoichiometric 0.5:1 PEG:protein feed, site-specific thiol-maleimide coupling yielded substantially higher conversion (∼81%) and a homogeneous single-species di-PEGylated (DoP 2) conjugate, whereas random amino-coupling produced heterogeneous mixtures across all PEG sizes (∼33–36% conversion, multiple degrees of polymerization). Purified single-species conjugates were benchmarked under a fixed IL-1β EC 80 challenge in HEK-Blue reporter cells; all retained full maximal efficacy but exhibited chemistry- and size-dependent right-shifts in IC 50, indicating steric modulation of receptor engagement rather than loss of function. To directly establish whether PEG attachment perturbs structural integrity, 2D 1 H– 15 N HSQC NMR on the 15 N-labeled thiol-PEG10 conjugate demonstrated a preserved global fold with localized chemical-shift perturbations near cysteine-proximal surface regions, confirming site-selective modification and intact binding scaffold. Together, these data establish the conjugation quality, antagonist activity, and structural integrity of the lead conjugate as the prerequisite characterization foundation for its further development and provide a transferable analytical framework for the rational design of PEGylated cytokine therapeutics.