İnterlökin-1 reseptör antagonisti için polimer konjugasyonunun optimizasyonu: verimlilik, ayrıştırılabilirlik ve fonksiyonel değerlendirme

This thesis builds a controlled, structure-guided comparison of PEGylation strategies for IL-1 receptor antagonist (IL-1Ra). Mapping accessible residues on IL-1Ra, thiol-selective (maleimide) versus lysine-directed (NHS) coupling strategies were evaluated across the same 5/10/20 kDa ladder at matched low stoichiometry, followed by a focused stoichiometry sweep for the preferred route. A near-native IL-1Ra backbone (M143V) was generated and 15N-labeled for HSQC NMR so that structural integrity and the specific impact of PEGylation could be evaluated independently of the marketed sequence. Across 5, 10, and 20 kDa, the thiol route gave a clean, predominantly di-PEG product, whereas lysine coupling produced heterogeneous mixtures across multiple sites and substitution levels. Increasing thiol stoichiometry above a low window reduced chromatographic separability without improving useful yield, identifying 10 kDa maleimide at low equivalents as a practical operating point. The M143V variant exhibited activity comparable to anakinra; 1H–15N HSQC of the 10 kDa thiol–PEG conjugate indicated preservation of a native-like fold with localized chemical-shift changes, and cell-based assays showed that PEGylation retained maximal inhibition while shifting the dose–response to higher concentrations. Together, the work delivers a reproducible analytical–structural–functional framework for selecting developable IL-1Ra conjugates, establishing a matched head-to-head design (chemistry × size × stoichiometry), and recommending thiol-selective 10 kDa PEG at low feed to obtain a clean di-adduct that purifies readily and preserves a native-like fold.