A single-step high-throughput bioassay for quantifying Fc-containing recombinant proteins based on non-classical calculation of fluorescence polarization

Abstract

The titer of recombinant proteins is one of the key parameters in biopharmaceutical manufacturing processes. The fluorescence polarization (FP)-based assay, a homogeneous, high-throughput and real-time analytical method, had emerged as a powerful tool for biochemical analysis and environmental monitoring. In this study, an FP-based bioassay was utilized to quantify antibody fragment crystallizable (Fc)-containing proteins, such as recombinant monoclonal antibodies (mAbs) and mAb derivatives, in the cell culture supernatant, and the impacts of tracer molecular weight and FITC-coupling conditions on fluorescence polarization were methodically examined. Distinct from the fluorescence polarization potency calculated by classical formula, we for the first time proposed a new concept and calculation of fluorescence polarization intensity, based on which an analytical method with broader detection range and analysis window was established for quantifying Fc-containing proteins. This provided new ideas for the practical application of fluorescence polarization theory. The established method could detect 96 samples within 30 minutes, with dynamic titer range of 2.5–400 mg L⁻¹, and a linear fitting R² between the measured and actual concentration reaching 0.99. The method had great application prospects in determining the titer of recombinant proteins with Fc fragments, especially when applied to large-scale screening of high-yield and stable expression CHO cell lines commonly used in biopharmaceutical industry.