mAb Titer Determination in 60 Seconds Using the Agilent Bio‑Monolith rProtein A Column

Significance of the topic

Rapid quantification of monoclonal antibody (mAb) titer is pivotal for high-throughput biopharma workflows such as clonal selection, process optimization, and QA/QC. The novel Agilent Bio-Monolith rProtein A column enables ultrafast affinity chromatography, reducing analysis time and increasing sample throughput.

Objectives and Overview

This study evaluates the performance of the Agilent Bio-Monolith rProtein A column at maximum flow rate and conducts a bridging comparison with the native protein A column. It demonstrates a 60-second chromatography method for mAb titer determination, suited for high-throughput clonal selection, process development, and optimization.

Instrumentation

• Agilent 1290 Infinity II Bio LC system: high-speed pump (G7132A), bio-inert pump (G5654A), multisampler (G7137A) with cooler (option 100), multicolumn thermostat with bio heat exchanger (G7116B), and diode array detector (G7117B) with bio flow cell.
• Analytical columns: Agilent Bio-Monolith rProtein A (4.95 × 5.2 mm, p/n 5190-6903) and native Bio-Monolith Protein A (4.95 × 5.2 mm, p/n 5069-3639).

Methodology

• Mobile phases: Eluent A (50 mM sodium phosphate, pH 7.4) and Eluent B (100 mM citric acid, pH 2.6).
• Gradient: binding at 0–0.2 min, elution at 0.3–0.65 min, reconditioning at 0.66–0.90 min.
• Flow rate: 3 mL/min; column temperature: 25 °C; detection: UV 280 nm; injection volume: 4 µL (10 µg loading).
• Samples: crude CHO cell culture supernatant containing 1 mg/mL recombinant IgG; calibration using purified mAb spiked into supernatant.

Main Results and Discussion

The rProtein A column achieved clear separation of host cell protein impurities (~0.05 min) and mAb (~0.61 min) across 60 consecutive injections, maintaining consistent peak shape and backpressure (~125 bar) and enabling ~60 samples/hour throughput. Calibration exhibited excellent linearity (R² = 0.9993). In the bridging study, rProtein A matched native Protein A in retention time, peak shape, linear response, sample carryover (none detected), and recovery. Recovery on rProtein A was ~1% lower than native but superior to other vendor columns.

Benefits and Practical Applications

• Ultrafast 60-second analysis significantly accelerates mAb titer determination for high-throughput screening and QA/QC.
• Direct replacement of native Protein A columns without performance loss simplifies method transfer.
• High linearity and minimal carryover ensure reliable quantification in demanding process development and manufacturing environments.

Future Trends and Opportunities

• Integration of rProtein A columns into multi-dimensional LC or automated platforms for real-time bioprocess monitoring.
• Extension to other antibody formats and biosimilar development for rapid titer screening.
• Adoption of inline analytics for closed-loop bioreactor control and accelerated process optimization.

Conclusion

The Agilent Bio-Monolith rProtein A column delivers robust, ultrafast, and high-throughput mAb titer determination, offering performance equivalent to native Protein A columns and supporting efficient biopharma process development, screening, and QC.

References

• Agilent Technologies. Application Note 5994-3969EN, August 2021.