Mathematical Simulations For Bioanalytical Assay Development: The (Un-)Necessity And (Im-)Possibility of Free Drug Quantification

Abstract

For every drug development program it needs to be discussed whether discrimination between free and total drug concentrations is required to accurately describe its pharmacokinetic behavior. This perspective describes the application of mathematical simulation approaches to guide this initial decision based on available knowledge about target biology, binding kinetics and expected drug concentrations. We provide generic calculations that can be used to estimate the necessity of free drug quantification for different drug molecules. In addition, mathematical approaches are used to simulate various assay conditions in bioanalytical ligand-binding assays: it is demonstrated that due to the noncovalent interaction between the binding partners and typical assay-related interferences in the equilibrium, a correct quantification of the free drug concentration is highly challenging and requires careful design of different assay procedure steps.

2017 White Paper on Recent Issues in Bioanalysis: A Global Perspective on Immunogenicity Guidelines & Biomarker Assay Performance (Part 3 – Lba: Immunogenicity, Biomarkers and PK Assays)

“…How can the biology and mechanism of action (MOA) help guide the development of the right assay? Use of 'ligand-free' matrix is a prerequisite for accurate 'free' drug quantification [15][16][17], but not fully covered in the current guideline, which focuses on 'total' drug quantification. What is the current practice by the industry?…”

“…For accurate free drug quantification and preparation of appropriate QC samples, the potential erroneous bias of the result due to the presence of soluble ligand in the matrix has to be considered. As described in references [15][16][17], critical steps are calibration and sample dilution. Use of soluble ligand-containing matrix for the calibration of a free drug assay might result in a systematic shift of the calibration curve and its use for sample dilution might result in the formation of new drug-ligand complexes since additional ligand is added to the equilibrium at every dilution step.…”

“…The relevance of the effects on the accurate free drug quantification requires a case specific evaluation, taking into consideration, among others, soluble ligand concentrations in the matrix as well as the assay sensitivity. The proposed use of 'ligand-free' matrix to circumvent any soluble target-related bias of the free drug assay result [16,17] was considered as a solution. However, the current practice in industry is still fragmented.…”

Factors That Impact Pharmacokinetic Measurements of Antibody Therapeutics: What is Your PK Assay Telling You?

“…The presence of these complexes can significantly influence drug/target complex equilibrium, depending on the choice of assay format as well as assay conditions such as incubation time and the extent of sample dilution. The assay conditions can in turn also impact the equilibrium in LBA between the drug and the assay reagents [8]. Due to the limited dynamic range of most assays, almost all samples (except for very low concentrations) have to be diluted in order to bring the drug concentration of the samples into the quantifiable calibration range.…”

“…Consequently the levels of free drug measured in an assay may not be representative of post-dose circulating levels in vivo. Another factor to be considered in free assays is a need for 'ligand-free' matrix for accurate free drug quantification [7][8][9]. Based on regulatory guidance, matrix effects should be evaluated by comparing calibration standard curves prepared in buffer to that prepared in matrix to demonstrate lack of matrix effect [10].…”