(156f) Continuous Capture of Monoclonal Antibody Breakthrough Using Immobilized Fluorescent Reporters in Downstream Processing Enhances Real-Time Bioseparation Efficiency

Efficient downstream processing is crucial in biopharmaceutical manufacturing, especially with advancements in cell culture technology leading to high antibody titers. A common initial step in downstream processing is the use of Protein A capture columns from Staphylococcus aureus (SpA), which selectively bind human IgG in complex cell culture fluid (CCF). However, real-time detection of antibody breakthrough in these UV-absorbing fluids is challenging, often resulting in underutilized column capacity and reduced productivity.
To enhance bioseparation efficiency, we have developed a fluorescence-based technology for continuous monitoring of antibody capture (1,2,3). This method employs reporters immobilized on CNBr-activated Sepharose 4B resin to detect IgG in clarified cell culture fluid. The column effluent is continuously contacted with immobilized fluorescein-labeled Fe-binding ligands, resulting in an immediately detectable fluorescence shift. This real-time monitoring allows for optimal use of column capacity, reducing waste and increasing productivity. Significant fluorescence shifts were detected at
0.5 g/L human IgG, enabling the detection of 5% breakthrough of a 10 g/L load within 2 minutes, with minimal interference from the sample matrix and negligible nonspecific protein binding.

Bibliography
1- A. Goyal et al., “Continuous monitoring of IgG using immobilized fluorescent reporters”, Biotechnol. Bioeng, vol. 120, no. 2, pp 482-490, 2023. https://doi.org/10.1002/bit.28254
2- U. Patil et al., “Continuous Fc detection for protein A capture process control”, Biosens. Bioelectron., vol. 165, no. 112327, 2020. https://doi.org/10.1016/j.bios.2020.112327
3- U. Patil et al., “Antibody Mix-and-Read Assays based on Fluorescence Intensity Probes”, MAbs, vol. 13, no. 1, 2021. https://doi.org/10.1080/19420862.2021.1980178