mAb Titer Analysis with the Agilent Bio-Monolith Protein A Column

Significance of the Topic

Monoclonal antibodies (mAbs) are central to modern therapeutics and require precise titer measurements during development. Accurate quantification of mAb concentration in cell culture supernatants underpins clone selection, process optimization and ensures consistent product quality.

Objectives and Overview of the Study

This study demonstrates how the Agilent Bio-Monolith Protein A column supports rapid and reliable determination of mAb titers. Using trastuzumab (Herceptin) as a model, the method is evaluated for precision, linearity, carryover and applicability to various CHO cell culture clones.

Methodology and Sample Preparation

Herceptin stock (21 mg/mL) was diluted in phosphate buffer for calibration; CHO supernatants were mixed 1:1 with 50 mM Na2HPO4 and centrifuged prior to injection. Chromatographic conditions included a 5.2 mm × 4.95 mm Bio-Monolith Protein A column with native Protein A monolith, a phosphate buffer (pH 7.4) for binding, and low pH elution using either 100 mM citric acid (pH 2.8) or acetic acid.

Used Instrumentation

• Agilent 1100 Series Quaternary Pump G1311A
• Agilent 1100 Series Autosampler G1313A
• Agilent 1100 Series Diode Array Detector G1315A
• Agilent OpenLAB CDS ChemStation C01.05

Key Results and Discussion

The monolithic format enabled sub-2-minute runs at up to 2 mL/min with minimal carryover. Precision tests showed peak area RSD <1.8% and retention time RSD <0.1% over 10 injections. The detection limit was 0.5 µg on-column, with linear calibration (R2 ≥ 0.9996) from 0.02 to 2 mg/mL. Comparing elution buffers, citric and acetic acid yielded comparable peak shapes and quantitation. Analysis of nine trastuzumab-producing CHO clones delivered consistent titer values enabling early high-producer selection.

Benefits and Practical Applications

The method is fast, robust and highly reproducible, demanding minimal sample prep and no extensive column cleaning. It is ideally suited for high-throughput mAb titer screening to accelerate clone selection and media optimization.

Future Trends and Opportunities

Future directions may include automation and integration with mass spectrometry for multi-attribute analysis, adaptation to novel antibody formats, and coupling with data-driven optimization workflows to further streamline biopharmaceutical development.

Conclusion

The Agilent Bio-Monolith Protein A column provides a powerful platform for rapid and accurate mAb titer determination in cell culture supernatants, supporting critical decision making in antibody development workflows.