Improving Peptide Mapping Studies and Reducing Assay Failures Through Reproducible Performance Using the ACQUITY Premier UPLC System (BSM)

Importance of the Topic

Liquid chromatography reproducibility is essential for reliable peptide mapping workflows, ensuring consistent peak assignment, accurate comparability across samples, and robust quality control in biopharmaceutical development.

Objectives and Study Overview

This study evaluates the performance of the ACQUITY Premier UPLC System configured with a Binary Solvent Manager (BSM) platform. Retention time reproducibility and peak tailing reduction by MaxPeak High Performance Surfaces (HPS) Technology were assessed across three systems and nine separation conditions to demonstrate assay robustness and transferability.

Materials and Methodology

The experiment utilized the Waters mAb Tryptic Digestion Standard (p/n 186009126) reconstituted in 300 µL of mobile phase A (0.1% formic acid in water). Key parameters included:

• LC configuration: ACQUITY Premier UPLC BSM with CSH C18 1.7 µm, 2.1×100 mm column at 60 °C, sample tray at 8 °C, 10 µL injection volume
• Mobile phases: 0.1% formic acid in water (A) and acetonitrile (B)
• Gradient programs: 5–95% B at 1%, 0.5%, and 0.25% B/min; flow rates of 0.5, 0.35, and 0.2 mL/min
• Detection: Tunable UV at 214 nm and ACQUITY QDa Mass Detector (positive mode, 350–1250 m/z)
• Data processing with Empower3 FR 4; metrics: retention time standard deviation and USP tailing factor on 18 representative peptides

Used Instrumentation

• ACQUITY Premier UPLC BSM System
• ACQUITY Premier Tunable UV Detector (10 mm flow cell, 214 nm)
• ACQUITY QDa Mass Detector
• QuanRecovery vials with MaxPeak HPS surface
• ACQUITY Premier CSH C18 Column (1.7 µm, 2.1×100 mm)
• Empower3 FR 4 chromatography data system

Key Results and Discussion

Retention time precision across three instruments and nine separation conditions yielded standard deviations below one second for most peptides. A Gaussian analysis indicated 68% of measurements within ±0.0057 min, 95% within ±0.0114 min, and 99.7% within ±0.0171 min. Chromatogram overlays under extreme gradient/flow combinations confirmed consistent performance without manual alignment. MaxPeak HPS surfaces reduced peak tailing by up to 83%, lowering variability and unveiling low-abundance deamidated species previously obscured.

Advantages and Practical Applications

• Exceptional inter- and intra-system retention time reproducibility
• Reduced nonspecific peptide adsorption and minimized peak tailing
• Enhanced sensitivity for low-abundance impurity detection
• Simplified method transfer and validation between laboratories
• Increased confidence in peptide attribute mapping and impurity assays

Future Trends and Potential Applications

Future developments may include integration with high-resolution mass spectrometry for deeper proteoform analysis, AI-driven method optimization, real-time bioprocess monitoring, and expansion of bio-inert surface technologies to a wider range of biotherapeutic assays.

Conclusion

The ACQUITY Premier UPLC BSM System delivers outstanding reproducibility and robustness in peptide mapping workflows. Combined with MaxPeak HPS surface technology, it minimizes analyte adsorption, enhances chromatographic performance, and supports reliable development and quality control of biopharmaceutical products.

References

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