Optimization of Incubation Time for Accurate Protein Binding Assessment in Biological Matrices Using Equilibrium Dialysis.

Authors

Jay Prakash Jain, Jianxia Shi, Atul Rathore, Austin Li, Chunyan Han, Xingjin Jiao, Richard Zang

Published in

Drug metabolism and bioanalysis. Volume 19. Issue 3. Pages 297-310.

Abstract

Accurate % unbound estimation in protein binding assays in drug discovery is critical for understanding Pharmacokinetics (PK), Pharmacodynamics (PD), and safety assessments. Short incubation times (2-6 hours) in Equilibrium Dialysis (ED) compromise the determination of the % unbound. Incubation times were evaluated in ED to obtain reliable estimates of % unbound across diverse biological matrices and compound types.
A time-course of 2-24 hours for 17 marketed compounds with varying physicochemical properties was conducted using RED and HTD devices across human plasma, tumor cell lines, 10% FBS, and rodent tissues to determine % unbound values.
Compounds exhibited unbound fractions ranging from <1% to >70% and generally correlated with LogD. Incubation times <6 hours underestimated % unbound (up to 6-fold), particularly for highly bound compounds. Equilibrium was typically reached by 16-24 hours in all matrices. A <20% increase was exhibited in % unbound beyond 16 hours (except lapatinib). HTD yielded a marginally faster equilibrium than RED.
Short incubation times (2-6 hours) in equilibrium dialysis often underestimate the % unbound, particularly for highly bound compounds. Extending incubation to ≥16 hours achieved equilibrium across plasma, tissues, and cell lines, yielding more reliable and consistent % unbound data. This standardized approach improves the PK/PD assessment and compound prioritization in early drug discovery.
The usual 2-6 hours of incubation in ED underestimates % unbound and may be inadequate for compound prioritization. We recommend ≥16 hours of incubation for protein binding assessment in early drug discovery.

PMID:
43016106
Bibliographic data and abstract were imported from PubMed on 12 Nov 2026.

Read full publication at:
Please sign in to see all details.

Stats

1-terrible, 9-excellent. How would you rate this publication? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.