Abstract
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 language | English (US) |
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Article number | 112327 |
Pages (from-to) | 112327 |
Journal | Biosensors and Bioelectronics |
Volume | 165 |
DOIs | |
State | Published - Oct 1 2020 |
Keywords
- Antibody breakthrough
- Fluorescence intensity
- Fluorescence polarization
- Process analytical technology
- Protein A chromatography
- Real-time monitoring
ASJC Scopus subject areas
- Biotechnology
- Biophysics
- Biomedical Engineering
- Electrochemistry