Comparative evaluation of ultrafiltration and EMSA formats for siRNA plasma protein binding: Methodological...

Plasma protein binding (PPB) is a critical pharmacokinetic parameter that modulates renal clearance and systemic safety of therapeutic small interfering RNAs (siRNAs), yet measurements vary significantly across methods. We compared ultrafiltration, agarose gel electrophoretic mobility shift assay (EMSA), and native polyacrylamide gel ESMA using the N-acetylgalactosamine (GalNAc)-conjugated siRNA, nedosiran, to identify method-dependent artifacts and guide reliable assessment. UF with real-time quantitative polymerase chain reaction across 0.5-25 μg/mL showed no concentration-dependent binding and an average unbound value of 4.21% ± 0.537%. Agarose gel EMSA (5-20 μg/mL) produced similar, concentration-independent results; incubation time and dilution sequence had negligible effects, but loading buffer composition and final salt altered the free fraction (higher salt results in more free concentration). Native polyacrylamide gel EMSA exhibited clear apparent concentration-dependent binding and was highly sensitive to dilution fold, salt concentration, and incubation time. Thus, EMSA outcomes are strongly assay-condition dependent and can produce misleading concentration effects unless conditions are tightly controlled. When nonspecific adsorption is minimized and device recovery is ensured, ultrafiltration with real-time reverse transcription quantitative polymerase chain reaction provides the most reliable PPB measurements for GalNAc-siRNAs. These findings clarify key methodological pitfalls and offer practical guidance for accurate GalNAc-siRNA PPB assessment. SIGNIFICANCE STATEMENT: Although general limitations of electrophoretic mobility shift assay have been reported previously, this study systematically quantifies the significant methodological divergence between agarose-based and PAGE-based electrophoretic mobility shift assay formats for small interfering RNA. By benchmarking against ultrafiltration, we reveal distinct artifact profiles for each gel matrix, providing critical guidance for interpreting discrepant plasma protein binding data in oligonucleotide development.