Demonstrating Method Equivalency of the ACQUITY UPLC H-Class PLUS Bio Binary System and an ACQUITY UPLC I-Class PLUS System Through Method Transfer of a Reversed-Phase Peptide Mapping Method

Significance of the Topic

Reliable transfer of chromatographic methods between legacy and modern LC platforms is essential in biopharmaceutical analysis to maintain data consistency, support regulatory compliance, and optimize resource utilization. Demonstrating method equivalency ensures that peptide mapping assays remain accurate and reproducible when instruments are upgraded or replaced.

Objectives and Study Overview

This study evaluated the equivalency of a reversed-phase liquid chromatography (RPLC) peptide mapping method transferred from an ACQUITY UPLC I-Class PLUS System to an ACQUITY UPLC H-Class PLUS Bio Binary System. Key performance metrics included retention time alignment, peak area reproducibility, and selectivity conservation across systems.

Methodology and Instrumentation

Peptide standard: reduced and alkylated tryptic digest of NIST mAb reference material at 0.5 µg/µL in 0.1% formic acid.
Chromatographic conditions: 2.1 × 100 mm, 1.7 µm BEH C18 column at 60 °C; flow rate 0.5 mL/min; injection volume 10 µL; column temperature 60 °C; sample tray at 10 °C; detection at 214 nm.
Gradient: linear elution from 3% to 40% acetonitrile (0.1% formic acid) over 30 min.
Data system: Empower 3 chromatography software with Gradient SmartStart Technology to compensate for dwell volume differences.

Used Instrumentation

• ACQUITY UPLC I-Class PLUS System (stainless steel flow path)
• ACQUITY UPLC H-Class PLUS Bio Binary System (biocompatible flow path)
• ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 µm, 2.1 × 100 mm
• QuanRecovery vials with MaxPeak HPS inserts
• Empower 3 FR4 chromatography software

Main Results and Discussion

• Retention time difference for 13 peptide peaks averaged 1.5 s between systems in default configurations, reflecting a dwell volume difference of ~12.5 µL.
• Intra-system retention time RSDs were below 0.002 min on both platforms, meeting specifications.
• Replacing the standard 50 µL mixer on the H-Class Bio with a 340 µL mixer introduced ~296 µL dwell volume shift; application of a 296 µL gradient delay via SmartStart realigned retention times without altering method parameters.
• Selectivity assessment yielded an R2 of 0.99996 for retention time correlation, confirming identical chromatographic profiles.
• Peak area percentage differences between systems were under 1% for all peptides, with most under 0.1%, demonstrating quantitative equivalency.

Benefits and Practical Applications of the Method

• Ensures seamless method migration between LC platforms in QC and contract laboratories.
• Maintains peptide mapping accuracy for complex biologic samples.
• Enables rapid compensation for hardware‐induced dwell volume differences via software adjustment rather than method revalidation.

Future Trends and Potential Applications

Advances in bioinert materials and fluidic design will further reduce surface interactions and dwell volume variability. Integration of automated gradient optimization and machine‐learning algorithms may streamline method transfer workflows. Expansion of standardized transfer protocols across additional LC technologies will support harmonized global analytics.

Conclusion

Method transfer from the ACQUITY UPLC I-Class PLUS to the H-Class PLUS Bio system preserved chromatographic selectivity, retention time precision, and quantitative accuracy. Gradient SmartStart Technology effectively compensated for dwell volume differences, enabling robust equivalency without method modification.

References

• Hong, P.; McConville, P.R. Dwell Volume and Extra-Column Volume: What Are They and How Do They Impact Method Transfer. Waters White Paper 720005723EN, May 2016.
• Dao, D.; Birdsall, R.E.; Yu, Y.Q. Instrument Mixer Considerations to Improve Assay Reproducibility and Sensitivity. Waters Application Note 720007011EN, September 2020.