Continuous monitoring of IgG using immobilized fluorescent reporters

Abstract In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real‐time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence‐based monitoring technology that allows batch mix‐and‐read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide‐activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein‐labeled Fc‐binding ligands in a small monitoring column to produce an immediately‐detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small‐scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber‐optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.