Critical evaluation of fluorescent dyes to evaluate the stability and ligand binding properties of an anti-cocaine mAb, h2E2

Abstract

Monoclonal antibodies have become a mainstay of modern drug development. However, unlike small molecule drugs, mAbs are large proteins that need to be characterized for their stability, heterogeneity, and tendency to aggregate. Many different extrinsic fluorescent dyes have been used to monitor the thermal stability, aggregation, and ligand binding characteristics of many different proteins. Some of these dyes change their fluorescence when bound to proteins due to changes in the hydrophobicity of their microenvironment (solvatochromic dyes such as Sypro Orange), while others respond to differences in rotational mobilities (rotor dyes such as DASPMI), and others have been used to detect fibrils and amyloid-like protein aggregation (amyloid dyes such as Thioflavin T). Previously, we used DASPMI dye and differential scanning fluorimetry to quantitate the binding of cocaine and cocaine metabolites to a humanized anti-cocaine h2E2 mAb under development for the treatment of cocaine use disorders. In the present study, we evaluated six dyes in these three classes for their ability to monitor domain denaturation and cocaine binding of the h2E2 mAb, both in its clinical formulation buffer and in PBS buffer. We noted that the Thioflavin T dye commonly used to assess amyloid formation was also capable of monitoring h2E2 mAb thermal denaturation and ligand binding using differential scanning calorimetry. However, unlike the DASPMI dye, the Thioflavin T dye caused a dose-dependent stabilization of the unliganded (apo) mAb, and when using the methodology developed with the DASPMI dye, decreased the apparent affinity of the mAb for cocaine as a function of dye concentration. Thus, although Thioflavin T differential fluorimetry data appears to be suitable for measuring cocaine affinity for this h2E2 mAb, the apparent mAb Kd values for cocaine are dependent on Thioflavin T dye concentration, reinforcing and extending the unique use of the DASPMI dye for this purpose.