Transferring Methods for Monoclonal Antibody Analysis from an Agilent 1260 Infinity Quaternary LC System to an ACQUITY Arc System

Significance of the Topic

Monoclonal antibodies are key biotherapeutics whose structural variants and purity must be accurately assessed.
Reliable transfer of chromatographic methods between labs and instruments minimizes revalidation efforts and ensures consistent results.

Study Objectives and Overview

This study evaluated the equivalency of size-exclusion chromatography (SEC) and peptide mapping methods transferred from an Agilent 1260 Infinity Quaternary LC system to a Waters ACQUITY Arc system.
Two platform methods—an isocratic SEC assay based on USP <129> and a gradient peptide map—were run without parameter changes to assess retention time, peak area, and resolution.

Methodology and Instrumentation

• Systems compared: Agilent 1260 Infinity Quaternary LC with HiP ALS and DAD; ACQUITY Arc with 2489 UV/Vis detector and Multi-flow path technology.
• SEC column: Tosoh TSKgel G3000SWxl, 250 Å, 5 µm, 7.8 × 300 mm; mobile phase: 0.2 M potassium phosphate, 0.25 M KCl, pH 6.2; flow: 0.5 mL/min.
• Peptide map column: XSelect CSH C18, 130 Å, 3.5 µm, 3 × 100 mm; mobile phases: water + 0.1% FA (A), acetonitrile + 0.1% FA (B); gradient with Gradient SmartStart; flow: 0.3 mL/min.
• Sample: rituximab at 10 mg/mL for SEC; tryptic digest at 0.5 mg/mL for peptide mapping.
• Data acquired at 280 nm (SEC) and 215 nm (peptide map); processed with Empower 3 CDS.

Main Results and Discussion

• SEC transfer: Retention times and peak area percentages for monomer, high and low molecular weight species differed by less than 0.3% and 0.07%, respectively.
• System precision (%RSD) for retention time and area remained below 2.0% on both systems; resolution and tailing met USP <621> criteria.
• Peptide map transfer: A 60 µL gradient offset was corrected using Gradient SmartStart; relative retention time differences for 20 peptides were <0.04.
• Overall chromatographic profiles, resolution, and reproducibility were conserved without method parameter adjustments.

Benefits and Practical Applications

• Streamlined method migration reduces time and costs associated with revalidation.
• Multi-flow path technology enables emulation of various LC platforms on a single system.
• Maintains regulatory compliance by meeting USP standards for monoclonal antibody analyses.

Future Trends and Potential Applications

• Expanded use of adaptive gradient control to automate dwell volume adjustments across diverse systems.
• Integration with advanced data analytics and AI-driven method optimization.
• Broader application to other biotherapeutics, including fusion proteins and novel modalities.

Conclusion

The ACQUITY Arc system demonstrated method equivalency with an Agilent 1260 Infinity system for both SEC and peptide mapping of rituximab, ensuring consistent performance and facilitating efficient method transfers in biopharmaceutical laboratories.

References

1. USP General Chapter <129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies, USP39-NF34 (2016)
2. USP General Chapter <621> Chromatography, USP38-NF33 (2015)
3. ACQUITY Arc System Brochure, Waters Corporation (2015)