Critical reagent generation, characterization, handling and storage workflows: impact on ligand binding assays.

The foundation of pharmacokinetics and antidrug antibodies assay robustness relies on the use of high-quality reagents. Over the past decade, there has been increasing interest within the pharmaceutical industry, as well as regulators, on defining best practices and scientific approaches for generation, characterization and handling of critical reagents. In this review, we will discuss current knowledge and practices on critical reagent workflows and state-of-the-art approaches for characterization, generation, stability and storage and how each of these steps can impact ligand-binding assay robustness.

Lay abstract A critical part of clinical development for new biologic drugs is the use of tests known as ligand-binding assay. These assays must be able to accurately measure drug levels and to assess if the biologic drug interacts with the immune system in patients. In order to support patient efficacy and safety, scientists must use state-of-the-art approaches to develop and identify specific reagents for each new biologic drug. This review aims to cover all key steps that are needed to support the quality and performance of the unique components of ligand-binding assays from the beginning of assay development and throughout the entire life-cycle of the biologic drug.

Using multiple platforms for critical reagents selection process to support pharmacokinetic ligand-binding assay development.

We have evaluated the utility of epitope binning on biolayer interferometry (BLI) as a strategy to funnel the selection of candidate pairs suitable for pharmacokinetic assay development. Totally, 8 anti-Idiotypic monoclonal antibodies in 64 possible combinations were tested by BLI, ELISA and Gyrolab®. Two epitope binning approaches were utilized, in-tandem and classic sandwich. Both formats identified four mutually exclusive bins providing 31 and 25 possible antibody pair combinations, respectively. In contrast, the ELISA and Gyrolab yielded 18 and 9 positive pairs, respectively, with only a partial correlation to the BLI results. Several positive pairs by ELISA and Gyrolab, screened negative by BLI. Just over half of the pairs predicted by BLI were positive on ELISA and less than a quarter were positive on Gyrolab. This evaluation showed, in our case, that BLI was limited in its ability to predict candidate pairs that would be successful in pharmacokinetic method development.

A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells.

Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.