Effects of Lipoprotein Binding and Plasma Dilution on the Accuracy of Ultracentrifugation Method in Plasma Protein Binding

Ultracentrifugation (UC) is the proposed methodology to study the protein binding of challenging compounds, such as targeted protein degraders, covalent inhibitors, and those with severe nonspecific binding properties. However, UC is prone to lipoprotein contamination that can result in artificially elevated fraction unbound (f_u) values compared to other methods. To quantitatively estimate the effect of binding to low-density lipoproteins/very-low density lipoproteins (LDL/VLDL) as well as plasma dilution on the data accuracy of ultracentrifugation method, a wide range of Log P(0.8-8.6), with or without report binding to LDL/VLDL, were selected, and the protein binding in human plasma was tested by the in house  UC protocol. The results obtained were compared with literature values tested on the dialysis-based method or the other methods.