How Shallow Can You Go? - Refining charge variant analysis of mAbs with the Agilent 1290 Infinity II Bio LC System

Importance of the topic

Charge variant analysis of monoclonal antibodies is a key quality control measure in biopharmaceutical development. Accurate separation of acidic and basic isoforms ensures consistent efficacy, safety, and regulatory compliance. Ultra-shallow salt gradients combined with robust instrumentation improve detection of subtle charge differences.

Objectives and study overview

This study evaluated salt gradient performance on the Agilent 1290 Infinity II Bio LC system for two model mAbs: trastuzumab and the NISTmAb reference standard. Key goals were to compare different gradient slopes for optimal resolution and to assess reproducibility under challenging shallow gradient conditions.

Methodology

Samples were prepared in 30 mM phosphate buffer at pH 6.8. Salt eluent consisted of phosphate buffer spiked with 500 mM NaCl. Salt gradient slopes ranged from 0.33 to 1 percent organic equivalent per minute (1.66 to 5 mM/min). Optimal pH was confirmed by scouting from 6.4 to 7.4, with pH 6.8 selected for both mAbs. Gradient development involved incrementally shallowing the slope to balance resolution against peak broadening.

Used instrumentation

• Agilent 1290 Infinity II Bio LC High-Speed Pump with iron-free flow path
• Agilent 1290 Infinity II Bio Multisampler with sample thermostat
• Agilent 1290 Infinity II Multicolumn Thermostat with biocompatible heat exchanger
• Agilent 1290 Infinity II Variable Wavelength Detector with biocompatible micro flow cell
• OpenLab CDS software version 2.5
• Bio MAb NP5 column 2.1 × 250 mm PEEK

Main results and discussion

Method development showed that very shallow gradients improved separation up to a point, beyond which peak broadening reduced practical resolution. The optimal slopes were 0.66 percent B/min (3.3 mM/min) for trastuzumab and 0.5 percent B/min (2.5 mM/min) for NISTmAb. Reproducibility studies over seven injections yielded retention time RSD below 0.06 percent and area RSD below 0.82 percent for major variants. Even an ultra-shallow gradient of 0.17 percent B/min (0.83 mM/min) delivered retention time precision below 0.25 percent RSD.

Benefits and practical applications

• High-confidence charge variant profiling for batch release and comparability studies
• Robust operation with corrosive buffers and extreme gradient conditions
• Improved separation of low-abundance charge isoforms
• Streamlined method development through precise solvent mixing

Future trends and opportunities

Advances in gradient control and integrated pH scouting promise further improvements in resolution and throughput. Coupling with mass spectrometry and automated data evaluation will enhance characterization of complex biotherapeutics. Expanded use in biosimilar comparability and next-generation antibody formats offers broad applicability.

Conclusion

The Agilent 1290 Infinity II Bio LC system, featuring an iron-free flow path and high-speed binary pump, delivers exceptional reproducibility and resolution in shallow salt gradient charge variant analysis. It represents a powerful tool for routine quality control and advanced biotherapeutic characterization.

Reference

• Dick LW Jr et al Identification and Measurement of Isoaspartic Acid Formation in the Complementarity Determining Region of a Fully Human Monoclonal Antibody J Chromatogr B 2009 877 3841–3849
• Zhang L et al Improving pH Gradient Cation-Exchange Chromatography of Monoclonal Antibodies by Controlling Ionic Strength J Chromatogr A 2013 1272 56–64
• Farnan D Moreno GT Multiproduct High-Resolution Monoclonal Antibody Charge Variant Separations by pH Gradient Ion-Exchange Chromatography Anal Chem 2009 81 8846–8857
• The LC Handbook Guide to LC Columns and Method Development Agilent Technologies publication 5990-7595EN 2016
• Goyon A et al Determination of Isoelectric Points and Relative Charge Variants of 23 Therapeutic Monoclonal Antibodies J Chromatogr B 2017 1065–1066 119–128
• Xie L et al Demonstrating Analytical Similarity of Trastuzumab Biosimilar HLX02 to Herceptin with a Panel of Sensitive and Orthogonal Methods Including a Novel FcγRIIIa Affinity Chromatography Technology BioDrugs 2020 34 363–379