In the manufacture of therapeutic monoclonal antibodies (mAbs), the
clarified cell culture fluid 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 IgG loading to avoid wastage.
Therefore, continuous real-time monitoring of IgG flowthrough is of
great interest. We previously developed a fluorescence-based monitoring
technology that allows mix-and-read mAb detection in cell culture fluid.
Here we report the use of reporters immobilized on CNBr-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 to produce an
immediately detectable change in fluorescence intensity. The technology
allows rapid and reliable monitoring of IgG in a flowing stream of
clarified cell culture fluid 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 2 minutes at a flow rate of 0.5