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Many tools for studying the pharmacokinetics of biologics lack single-cell resolution to quantify the heterogeneous tissue distribution and subsequent therapeutic degradation in vivo. This protocol describes a dual-labeling technique using two near-infrared dyes with widely differing residualization rates to efficiently quantify in vivo therapeutic protein distribution and degradation rates at the single cell level (number of proteins/cell) via ex vivo flow cytometry and histology. Examples are shown for four biologics with varying rates of receptor internalization and degradation and a secondary dye pair for use in systems with lower receptor expression. Organ biodistribution, tissue-level confocal microscopy, and cellular-level flow cytometry were used to image the multi-scale distribution of these agents in tumor xenograft mouse models. The single-cell measurements reveal highly heterogeneous delivery, and degradation results show the delay between peak tumor uptake and maximum protein degradation. This approach has broad applicability in tracking the tissue and cellular distribution of protein therapeutics for drug development and dose determination.

Practical Guide for Quantification of In Vivo Degradation Rates for Therapeutic Proteins with Single-Cell Resolution Using Fluorescence Ratio Imaging