A framework for plasma protein binding: Comparing methods for diverse small molecules and adapting for novel...

Reliable measurements of plasma protein binding (PPB) are essential for determining a drug's therapeutic window and understanding pharmacokinetic/pharmacodynamic (PK/PD) relationships. This study evaluates the concordance of the three primary PPB methods-equilibrium dialysis (ED), ultrafiltration (UF), and ultracentrifugation (UC)-using an orthogonal approach across 26 small molecules with diverse physicochemical properties. Results demonstrated strong correlation between methods (R² = 0.95), with ED data aligning closely with literature values (R² = 0.97). While ED remains the preferred high-throughput method, its limitations for "beyond Rule of 5″ (bRo5) and highly bound compounds were addressed through strategic method adaptations and exploring Transil as an alternative method. For modalities including both linear and cyclic peptides as well as degraders, methods were optimized to address specific challenges. For peptides, ED incubation times were increased from 5 h to 16 h, and the addition of 0.01% Solutol® was used to mitigate non-specific binding (NSB), with orthogonal validation using an adapted UC method (using 50% diluted plasma) providing high confidence in results (R² > 0.87). For degraders, standard high-throughput dialysis (HTD) remained suitable, where species-dependent low recovery successfully served as a predictor for enzymatic plasma instability rather than NSB. A decision tree is introduced to guide method selection based on compound modality and challenges such as stability and solubility, ensuring robust PPB assessment for both traditional and novel therapeutic chemical classes.