TY - JOUR
T1 - Continuous Fc detection for protein A capture process control
AU - Patil, Ujwal
AU - Crum, Mary
AU - Vu, Binh
AU - Wasden, Katherine
AU - Kourentzi, Katerina
AU - Willson, Richard C.
N1 - Funding Information:
We would like to thank Dr. Glen Bolton (Amgen), Dr. David Robbins (AstraZeneca), Dr. David Roush (Merck Research Labs), Dr. Kent Göklen (GlaxoSmithKline), and Dr. John Erickson (National Institute for Innovation in Manufacturing Biopharmaceuticals) for valuable discussions. Special thanks to Golden LEAF Biomanufacturing Training and Education Center (BTEC), North Carolina State University for generously donating CHO cell culture fluid used in this study. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Publisher Copyright:
© 2020 Elsevier B.V.
PY - 2020/10/1
Y1 - 2020/10/1
N2 - 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.
AB - 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.
KW - Antibody breakthrough
KW - Fluorescence intensity
KW - Fluorescence polarization
KW - Process analytical technology
KW - Protein A chromatography
KW - Real-time monitoring
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U2 - 10.1016/j.bios.2020.112327
DO - 10.1016/j.bios.2020.112327
M3 - Article
C2 - 32729475
AN - SCOPUS:85087418248
VL - 165
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
SN - 0956-5663
M1 - 112327
ER -