Continuous Fc detection for protein A capture process control

Ujwal Patil, Mary Crum, Binh Vu, Katherine Wasden, Katerina Kourentzi, Richard C. Willson

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


Purification of therapeutic monoclonal antibodies usually involves a protein A affinity capture step. Because column breakthrough of antibody in complex, UV-absorbing culture fluid cannot be readily detected in real time, processes are designed so conservatively that column capacity is usually underutilized, wasting adsorbent and reducing productivity. We have developed a fluorescence-based monitoring technology which allows real-time mAb monitoring and used it to detect IgG in column breakthrough. The column effluent was continuously contacted with soluble fluorescein-labeled Fc-binding ligands to produce an immediately-detectable shift in both fluorescence polarization and intensity. To extend the upper limit of inlet flow rate, a 14:1 split-ratio flow splitter was tested with an inlet flow of 15 mL/min (0.9 L/h), producing a sampling stream at 1 mL/min while still enabling continuous detection functionality. We observed significant shifts in fluorescence intensity in CHO cell culture fluid spiked with human IgG, and detected 0.02–0.1 g/L human IgG in protein A column breakthrough at a flow velocity of 80 cm/h. The increase in fluorescence intensity upon 0.1% breakthrough of an 1 g/L feed was used to trigger column switching using Python-enabled two-way communication with the standard Unicorn OPC process control protocol. The technology allows rapid, continuous and reliable monitoring of IgG in a flowing process stream, without elaborate sample preparation.

Original languageEnglish (US)
Article number112327
Pages (from-to)112327
JournalBiosensors and Bioelectronics
StatePublished - Oct 1 2020


  • Antibody breakthrough
  • Fluorescence intensity
  • Fluorescence polarization
  • Process analytical technology
  • Protein A chromatography
  • Real-time monitoring

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry


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