High-throughput protein binding assay using cross-sample pooling in combination with high-resolution mass...

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Analytical Chemistry
• Biotechnology

Background:

• The unbound fraction of drugs (ƒ_u) is critical for understanding drug disposition, efficacy, and toxicity.
• Accurate measurement of ƒ_u is essential in drug development, driving demand for efficient and reliable assays.
• Current protein binding assays are resource-intensive, necessitating innovative approaches to improve throughput and reduce consumption.

Purpose of the Study:

• To develop and validate novel cross-pooling and cassetting procedures for protein binding assays.
• To enhance assay throughput and comprehensiveness while minimizing biological and experimental resource utilization.
• To leverage high-resolution mass spectrometry (HRMS) for rapid and informative analysis of unbound drug fractions.

Main Methods:

• Implementation of a cross-pooling strategy where samples are equalized with complementary matrices from different drugs.
• Integration of sample cassetting to further increase assay throughput.
• Analysis of samples using high-performance liquid chromatography coupled with Orbitrap HRMS for accurate ƒ_u determination.

Main Results:

• Highly comparable human plasma ƒ_u values were obtained for 27 diverse drugs using cross-pooling versus conventional methods.
• Validation of the cross-pooling method across different species (rat, mouse) and matrices (microsomes, brain).
• HRMS demonstrated consistent quantification comparable to triple quadrupole analysis, with added advantages in throughput and versatility.

Conclusions:

• The combined cross-pooling, cassetting, and HRMS approach is experimentally and analytically feasible.
• This method offers up to an 8-fold increase in throughput and a 50% reduction in matrix consumption.
• The developed assay is resource-effective, analytically dependable, and versatile for various drug development applications.