Assessment of Free Dye in Solutions of Dual-Labeled Antibody Conjugates for In Vivo Molecular Imaging

Recent preclinical and clinical studies show that dyes that excite and fluoresce in the near-infrared range may be used for tracking and detecting disease targets in vivo. A method for quantifying free dye molecules in antibody conjugate preparations is required for agent batch release and for translation into the clinic. Herein, we developed and validated a SDS-PAGE method to determine the percentage of free IRDye 800CW in (DTPA)(n)-trastuzumab-(IRDye 800)(m) conjugate sample preparations in which high-performance liquid chromatography (HPLC) assessment of free dye was not possible. The SDS-PAGE assay was accurate and valid for free IRDye 800CW amounts between 38 and 4 mol% of total dye. Gel sample preparation reagent affected the specificity of the assay, and lower and upper limits of quantitation and detection were determined. This method may be applicable to other near-infrared dye-conjugated antibody-based imaging agents in which HPLC assessment of purity is not feasible. This validated method for quality assurance will facilitate the translation of dual-labeled antibody conjugates for nuclear and optical imaging.