Impact of Instrument Characteristics on Reversed Phase Chromatography Method Transfer Across Biocompatible UHPLC Systems

Importance of the Topic

Reliable transfer of reversed-phase chromatography methods across biocompatible UHPLC systems is essential for efficient biopharmaceutical analysis. Consistent performance between instruments reduces method redevelopment, minimizes validation burden, and ensures data comparability across internal laboratories and external contract research organizations.

Objectives and Study Overview

This study evaluates the transferability of a reversed-phase (RP) separations method for monoclonal antibody (mAb) subunits between two bio-inert UHPLC platforms: the Waters ACQUITY Arc Bio System and the Agilent 1260 Infinity Bio-inert System. Key metrics such as retention time, peak area percentage, peak width, extra-column volume, and dwell volume were compared to assess method performance consistency.

Methodology and Instrumentation

A reduced, IdeS-digested NIST mAb standard was analyzed under identical chromatographic conditions on both systems. Applied Instrumentation:

• Waters ACQUITY Arc Bio System: QSM-R Bio pump (paths 1 and 2), FTN-R Bio autosampler, 2998 PDA detector.
• Agilent 1260 Infinity Bio-inert System: 1260 Bio Quat pump, 1260 HiP Bio ALS autosampler, 1260 DAD VL+ detector.
• Column: BioResolve RP mAb Polyphenyl, 450 Å pore, 2.7 µm, 4.6 × 50 mm at 65 °C.
• Mobile phases: 0.1% TFA in water (A) and in acetonitrile (B); flow 0.96 mL/min; injection 19.2 µL; detection at 280 nm; gradient from 85% to 20% A over 21.3 min.

Main Results and Discussion

Measured extra-column volumes were comparable (19 µL vs. 24 µL), yielding similar peak widths. Dwell volumes were 1.32 mL (Arc path 1), 1.01 mL (Arc path 2), and 1.57 mL (Agilent). Retention time differences averaged 0.07 min (<1% shift) across the two systems. Peak area percentages showed <1% absolute difference and %RSDs below 1%, confirming reproducible quantitation. The Arc system’s Multi-flow path Technology and Gradient SmartStart effectively compensated for dwell volume disparities without modifying the gradient table.

Benefits and Practical Applications

• Streamlined method transfers reduce development time and resource expenditure.
• Maintains validated workflows and regulatory compliance by avoiding manual gradient edits.
• Ensures consistent quantitation of mAb subunits across multi-vendor platforms for QA/QC and research.

Future Trends and Potential Applications

Advances in modular flow-path architectures and software-based gradient delay compensation will further simplify cross-platform method portability. Emerging instrument designs aimed at reducing extra-column effects will support high-throughput characterization of complex biotherapeutics and facilitate global method standardization.

Conclusion

The RP separation of mAb subunits can be successfully transferred between biocompatible UHPLC systems with minimal performance drift. Matching extra-column and dwell volumes, combined with Multi-flow path Technology and Gradient SmartStart, enables reproducible retention times and quantitation without manual gradient adjustments.

Reference

1. Zhang X. et al. High-Throughput Analysis of Antibody Subunits Using a BioResolve RP mAb Polyphenyl Column. Waters Application Note 720006204EN, Jan 2018.
2. Waters Care and Use Manual. mAb Subunit Standard. 720006154EN.
3. Hong P., McConville P.R. Dwell Volume and Extra-Column Volume: Impact on Method Transfer. Waters Application Note 720005723EN, 2016.
4. Chapter 621 Chromatography. United States Pharmacopeia and National Formulary (USP 37-NF 32 S2), 2014.