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Continuous Monitoring of IgG using Immobilized Fluorescent Reporters
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  • Atul Goyal,
  • Binh Vu,
  • Vijay Maranholkar,
  • Ujwal Patil,
  • Katerina Kourentzi,
  • Richard Willson
Atul Goyal
University of Houston

Corresponding Author:[email protected]

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Binh Vu
University of Houston
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Vijay Maranholkar
University of Houston
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Ujwal Patil
University of Houston System
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Katerina Kourentzi
University of Houston
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Richard Willson
University of Houston
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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.
23 Dec 2021Submitted to Biotechnology and Bioengineering
24 Dec 2021Submission Checks Completed
24 Dec 2021Assigned to Editor
04 Jan 2022Reviewer(s) Assigned
20 Mar 2022Review(s) Completed, Editorial Evaluation Pending
20 Mar 2022Editorial Decision: Revise Major
23 Sep 20221st Revision Received
01 Oct 2022Submission Checks Completed
01 Oct 2022Assigned to Editor
08 Oct 2022Review(s) Completed, Editorial Evaluation Pending
08 Oct 2022Editorial Decision: Accept