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 aprotein 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.
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
mL/min.
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