Continuous monitoring of IgG using immobilized fluorescent reporters

Atul Goyal, Binh Vu, Vijay Maranholkar, Ujwal Patil, Katerina Kourentzi, Richard C. Willson

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)482-490
Number of pages9
JournalBiotechnology and Bioengineering
Issue number2
StatePublished - Feb 2023


  • antibody breakthrough
  • fluorescence intensity
  • online monitoring
  • process analytical technology
  • protein A chromatography
  • Chromatography, Affinity
  • Humans
  • Immunoglobulin G
  • Coloring Agents
  • Ligands
  • Antibodies, Monoclonal
  • Staphylococcal Protein A

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Bioengineering
  • Biotechnology


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