Demonstrating the Applicability of the ACQUITY™ Premier Binary System for Long Shallow Gradient Peptide Mapping Analysis

Importance of the Topic

Reversed-phase liquid chromatography (RPLC) is an essential tool for mapping peptides generated from the enzymatic digestion of complex proteins such as monoclonal antibodies. Achieving high resolution of closely related peptide variants often requires long, shallow organic gradients, which in turn demand exceptional gradient fidelity to maintain consistent retention times. Reliable retention time reproducibility is critical for accurate peptide identification, method comparability, and quality control in biopharmaceutical analysis.

Goals and Study Overview

This study evaluates the performance of the Waters ACQUITY Premier Binary System with MaxPeak HPS Technology against a leading biocompatible UHPLC binary system (Competitor X). Over a five-day period, both systems were assessed for retention time repeatability of 21 peptides from an enolase digestion standard using identical columns and mobile phases under long, shallow gradient conditions.

Methodology and Instrumentation

Sample preparation and chromatography were carried out as follows:

• MassPREP Enolase Digestion Standard reconstituted in 0.1% TFA, vortex-mixed, and analyzed from chilled autosampler vials.
• ACQUITY Premier and Competitor X systems run in parallel with new columns at study start; dynamic leak tests and system qualification performed daily.
• Column: ACQUITY UPLC Peptide CSH C18, 130 Å, 1.7 µm, 2.1 × 150 mm at 65 °C; sample temperature 6 °C.
• Gradient: 1% to ~50% acetonitrile over 85 minutes (0.58% B per minute) with 0.1% TFA modifiers.
• Detection: Photodiode array at 214 nm, 10 Hz; data collected in Empower 3.6.1.

Used Instrumentation

• Waters ACQUITY Premier UPLC Binary System with MaxPeak™ HPS Technology and APC Reservoir Cap Kit
• Competitor X Biocompatible UHPLC Binary System
• ACQUITY UPLC Peptide CSH C18 Column, 130 Å, 1.7 µm, 2.1 × 150 mm
• Photodiode Array Detector / Diode Array Detector (214 nm)
• Empower™ 3.6.1 Chromatography Data Software

Main Results and Discussion

Both systems delivered high-quality peptide separations under long, shallow gradient conditions, with all 21 monitored peaks exhibiting five-day retention time standard deviations below 0.045 minutes. The ACQUITY Premier System achieved an average standard deviation of 0.017 minutes versus 0.030 minutes for the competitor, representing 13–146% improved repeatability across individual peptides. Slight shifts in absolute retention times between systems were attributed to differences in system volume, gradient delay, and selectivity, underscoring the importance of precise pump performance for method transfer.

Benefits and Practical Applications

• Superior retention time repeatability for long, shallow gradients in peptide mapping workflows
• Enhanced gradient fidelity simplifies comparability of peptide maps without complex alignment
• Supports robust ultraviolet and mass spectrometric detection with reduced variability

Future Trends and Potential Applications

Ongoing advancements in binary pump technologies will further refine gradient precision and system robustness. Integration with high-resolution mass spectrometry, automation of method transfer using AI-driven algorithms, and adoption of high-throughput, miniaturized workflows are expected to expand the applicability of RPLC peptide mapping in biopharmaceutical development and quality control.

Conclusion

The ACQUITY Premier Binary System demonstrates superior gradient delivery precision and retention time reproducibility compared to a competitive UHPLC binary system under long, shallow gradient conditions. Its performance makes it an ideal platform for detailed peptide mapping of biotherapeutic proteins, enhancing method reliability and data comparability in regulated environments.

References

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4. Simeone, J.; Hong, P.; McConville, P. R. Performance of the ACQUITY UPLC I-Class PLUS System for Methods Which Employ Long, Shallow Gradients. Waters Application Note 720006290, 2018.