AUTHOREA
Log in
Sign Up
Browse Preprints
LOG IN
SIGN UP
Essential Site Maintenance
: Authorea-powered sites will be updated circa 15:00-17:00 Eastern on Tuesday 5 November.
There should be no interruption to normal services, but please contact us at
[email protected]
in case you face any issues.
Binh Vu
Public Documents
1
Continuous Monitoring of IgG using Immobilized Fluorescent Reporters
Atul Goyal
and 5 more
January 04, 2022
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.