Transfer of an SEC Method for Monoclonal Antibody Analysis from HPLC to UHPLC Using the ACQUITY Arc System

Importance of the Topic

Size exclusion chromatography (SEC) is vital for monitoring aggregation and quality of monoclonal antibodies throughout development and manufacture. Reliable transfer of SEC methods between labs ensures consistent product assessment and regulatory compliance.

Objectives and Study Overview

The study aimed to evaluate method transfer of a USP-derived SEC protocol for rituximab from an Agilent 1100 HPLC to the Waters ACQUITY Arc System and then upgrade to UHPLC conditions. Metrics of retention time, peak area and resolution were used to demonstrate equivalency and performance gains.

Methodology and Instrumentation

• Sample: Rituximab at 1 mg/mL in 0.02 M sodium phosphate, 0.3 M NaCl, pH 6.8
• HPLC setup: Agilent 1100 Series with quaternary pump, DAD, Tosoh TSK gel G3000 SW XL column (7.8×300 mm, 5 μm), 0.5 mL/min, 30 °C, detection at 280 nm, 35 min run
• SEC-HPLC transfer: ACQUITY Arc System with 2489 UV/Vis detector under identical conditions
• UHPLC update: ACQUITY Arc with XBridge Protein BEH SEC column (7.8×300 mm, 3.5 μm), flow rate 0.714 mL/min, run time 24.5 min
• Data management: Empower 3 CDS

Main Results and Discussion

• Retention times matched within 0.2 min and monomer peak areas differed by <0.02% after transfer
• Five-injection repeatability on the Arc System yielded %RSD <0.06% for retention time and peak area
• UHPLC conditions improved dimer-monomer resolution from 1.52 to 1.75, with sharper peaks and 30% shorter run time
• Consistent peak area percentages confirmed minimal SEC interaction effects

Benefits and Practical Applications

The Arc System allows seamless replication of HPLC methods and straightforward adoption of UHPLC, boosting lab productivity through faster separations and better resolution without method revalidation.

Future Trends and Opportunities

• Wider use of multi-flow path platforms to unify HPLC/UHPLC workflows
• Development of high-throughput SEC-UHPLC assays for biotherapeutic screening
• Integration with advanced detectors and automated data analysis for real-time aggregation monitoring

Conclusion

SEC method transfer from Agilent 1100 to ACQUITY Arc was successful, and upgrading to UHPLC delivered enhanced resolution and reduced run time while maintaining analytical consistency.

Reference

1. ACQUITY Arc System Brochure, Waters Corporation, 2015
2. U.S. Pharmacopeial Convention, USP<621> Chromatography, USP38-NF33, 2015