The titer of recombinant proteins is one of the key parameters in foundational biopharmaceutical manufacturing processes. The fluorescence polarization (FP) based assay, a completely homogeneous, high-throughput, and real-time analytical method, has emerged as a powerful tool for biochemical analysis and environmental monitoring. In this study, a FP-based bioassay was utilized to quantify Fc-containing proteins in cell culture supernatant, and the impact of tracer molecular weight and FITC coupling conditions on fluorescence polarization was methodically examined. Innovatively, distinct from the classical normalization calculation formula, we first proposed a new concept of fluorescence polarization intensity. An analytical method was established based on this concept, offering a wide detection range and analysis window for the quantification of Fc-containing proteins. This provides new ideas for the practical application of fluorescence polarization theory. The established method could detect 96 samples within 30 minutes, with a dynamic titer range of 2.5-400 mg/L, and a linear fitting R2 between the measured and actual concentration reaching 0.99. The method has 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 cell lines in cell line development.

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