Combining small-scale purification and analysis of monoclonal antibodies on one instrument

Importance of the Topic

Monoclonal antibodies are central to modern therapeutics and require rigorous purification and characterization to ensure safety, efficacy, and regulatory compliance. Integrating preparative purification and detailed analysis into a single workflow accelerates development and improves laboratory efficiency.

Goals and Study Overview

This study demonstrates a unified workflow for small-scale purification of an anti-c-Myc monoclonal antibody spiked into 5 mL of E. coli lysate, followed by polishing and comprehensive analysis on one Agilent 1260 Infinity Bio-inert Quaternary LC System.

Methods and Instrumentation

The system comprised a bio-inert quaternary pump, high-performance autosampler, two thermostats, column compartment with solvent heat exchanger, diode-array detector, manual injector with 20 mL PEEK loops, and fraction collector. Key columns included:

• HiTrap Protein A HP (1 mL) for affinity capture
• Superdex 200 10/300 GL (10 × 300 mm) for size exclusion polishing
• Agilent Protein A Monolith (4.6 × 250 mm) for identification and quantification
• Bio MAb weak cation exchange column (4.6 × 250 mm) for charge variant analysis

OpenLAB CDS ChemStation controlled the runs and Agilent Buffer Advisor designed a four-component ionic strength gradient at pH 6.4. Samples were prepared by spiking purified anti-c-Myc into lysate and concentrating fractions via 30 kDa ultrafiltration.

Main Results and Discussion

• The Protein A capture step achieved selective binding of the antibody from >100 mg/mL total protein lysate at 0.5 mL/min, with system backpressure <5 bar.
• SEC polishing resolved host protein contaminants at low flow and <10 bar, yielding five fractions; only the main peak contained antibody as confirmed by Protein A monolith analysis.
• Quantification using a calibration curve (78 ng–20 µg) delivered R²=0.99874 and a final antibody concentration of 0.94 mg/mL.
• Charge variant profiling revealed minor acidic and basic species matching the reference antibody profile, indicating preservation of molecular integrity.

Benefits and Practical Applications

• Consolidates preparative and analytical steps on one instrument, reducing sample transfers and contamination risk.
• High-volume injections with bio-inert PEEK components support preparative loads without metal contact.
• Low system backpressure prolongs column lifetime and allows flexible method development.
• Rapid turnaround supports workflows in biopharma R&D, QA/QC, and process development.

Future Trends and Opportunities

Future developments may include inline concentration modules, integration with mass spectrometry for structural analysis, expanded software-guided gradient optimization, and application to a broader range of biotherapeutics such as fusion proteins and ADCs.

Conclusion

The Agilent 1260 Infinity Bio-inert Quaternary LC enables efficient small-scale purification and thorough analytical characterization of monoclonal antibodies on a single platform, streamlining workflows and ensuring high-quality biotherapeutic production.

Reference

• Low D. et al. Future of antibody purification. Journal of Chromatography B, 848:48–63 (2007).
• Protein Purification Handbook. GE Healthcare.