Method Transfer of Size-Exclusion and Ion-Exchange Methods for Monoclonal Antibody Analysis from HPLC to the Arc Premier System

Significance of the Topic

Size‐exclusion chromatography (SEC) and ion‐exchange chromatography (IEX) are cornerstone techniques in the quality control of monoclonal antibodies. Robust method transfer between laboratories and modernization of liquid chromatography (LC) platforms ensure consistent data generation throughout biopharmaceutical product lifecycles.

Study Objectives and Overview

This work evaluated the transfer of legacy SEC and IEX methods from an Alliance HPLC System to the Arc Premier UHPLC System. Key goals included demonstrating method equivalency, assessing chromatographic performance, and exploring the benefits of modern column chemistries.

Methodology and Instrumentation

Sample Preparation

• Trastuzumab at 10 mg/mL for SEC in phosphate buffer with KCl (pH 6.2).
• Trastuzumab at 1 mg/mL for IEX in MES buffer (pH 6.6) with NaCl gradient.

Instrumentation Used

• Alliance HPLC System with 2489 UV/Visible Detector.
• Arc Premier UHPLC System with 2489 UV/Visible Detector using MaxPeak HPS technology.
• Empower 3 Chromatography Data Software FR4.

Chromatographic Conditions

• SEC columns: BioSuite Diol (5 µm) and BioResolve SEC mAb (2.5 µm).
• IEX columns: Protein‐Pak Hi Res CM/SP (7 µm) and BioResolve SCX mAb (3 µm).
• SEC isocratic run, 30 min at 0.5 mL/min; IEX salt gradient at 0.8 mL/min, 25 °C.

Results and Discussion

Method Equivalency

• SEC: Retention time differences between platforms were < 0.6 min; peak area differences < 0.07 %.
• IEX: Retention time shifts matched dwell‐volume differences; variant peak areas agreed within 0.1 %.
• Repeatability: %RSD for all peaks < 2 % on each system.

Modern Column Chemistries

• SEC: Smaller‐particle BioResolve column resolved low‐molecular‐weight fragment (LMWS 1) previously co‐eluting with the monomer, revealing ~0.6 % monomer overestimation in legacy methods.
• IEX: Strong cation‐exchange BioResolve SCX mAb column resolved the greatest number of charge variants compared to 7 µm CM and SP columns under identical conditions.

Benefits and Practical Applications

• Verified that Arc Premier UHPLC supports existing HPLC‐based SEC and IEX assays without compromising data comparability.
• Enhanced resolution and quantitation of low‐abundance species and charge variants through updated column chemistries.
• Reduced run times and improved method robustness, aiding high‐throughput quality control workflows.

Future Trends and Applications

Laboratories will increasingly adopt UHPLC platforms with advanced surface technologies to minimize analyte‐surface interactions. Emerging opportunities include:

• Integration of multi‐attribute methods combining SEC, IEX, and MS detection.
• Automation and AI‐driven method optimization for rapid screening of column and mobile‐phase combinations.
• Continued development of tailored stationary phases for enhanced selectivity in biotherapeutic analysis.

Conclusion

The Arc Premier UHPLC System successfully transferred legacy SEC and IEX methods from a standard HPLC platform, exhibiting negligible differences in retention time and peak area. Coupling modern UHPLC columns with this system delivers superior resolution, faster analyses, and future‐proofs biopharmaceutical laboratories against evolving analytical demands.

References

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