Plasma protein binding methods (ED, UF, UC) show strong correlation, optimized for linear/cyclic peptides and degraders

Plasma protein binding (PPB) is crucial for defining a drug's therapeutic window and understanding pharmacokinetic/pharmacodynamic (PK/PD) relationships. Traditional PPB methods often fall short for "beyond Rule of 5" (bRo5) compounds, highly bound molecules, and novel modalities like peptides and degraders due to challenges like non-specific binding or stability. This study addresses these gaps by evaluating existing methods and developing optimized protocols for these complex therapeutic classes.

Researchers evaluated the concordance of three primary PPB methods: equilibrium dialysis (ED), ultrafiltration (UF), and ultracentrifugation (UC). An orthogonal approach was used across 26 small molecules with diverse physicochemical properties. For novel modalities, methods were optimized: for peptides, ED incubation was increased from 5h to 16h, and 0.01% solutol was added to mitigate non-specific binding (NSB). Orthogonal validation for peptides used an adapted UC method with 50% diluted plasma. For degraders, standard high-throughput dialysis (HTD) was assessed.

The study demonstrated a strong correlation between the three primary PPB methods (R2 = 0.95). ED data showed close alignment with literature values (R2 = 0.97).

For peptides, the adapted ED method (increased incubation, 0.01% solutol) and orthogonal UC validation (using 50% diluted plasma) provided high confidence in results, with R2 > 0.87. For degraders, standard HTD proved suitable, and species-dependent low recovery successfully predicted enzymatic plasma instability rather than NSB. A decision tree was introduced to guide method selection based on compound modality, stability, and solubility challenges.