DEPLOYING EFFICIENT WORKFLOWS FOR PEPTIDE MAPPING WITH LC-UV/MS PLATFORMS

Importance of the topic

Peptide mapping by liquid chromatography with UV and mass spectrometric detection is a cornerstone analytical approach in biopharmaceutical development and manufacturing. It enables detailed characterization of protein therapeutics, identification of post-translational modifications and stress-induced variants, and supports quality control from early-stage process design through commercial release. Automating these workflows enhances throughput, reproducibility and ease of technology transfer across laboratories.

Objectives and overview

The study aimed to deploy and evaluate an efficient, end-to-end peptide mapping workflow using an ACQUITY Premier LC-UV/MS platform integrated with Empower software. Key goals included assessing inter- and intra-system reproducibility of retention times under varying chromatographic conditions, demonstrating the performance of MaxPeak High-Performance Surface (HPS) technology for peak shape improvement, and validating a universal automated data-processing method for both upstream and downstream peptide mapping applications.

Methodology

Monoclonal antibody (mAb) digestion samples were injected in triplicate on three ACQUITY Premier Binary Solvent Manager (BSM) systems. Nine distinct gradient and flow-rate conditions challenged the binary pump performance. A stress study of infliximab was also performed to monitor degradation attributes over time.

Used instrumentation

• ACQUITY Premier BSM LC System
• ACQUITY Premier CSH C18 column
• Thermal UV (TUV) detector
• ACQUITY QDa mass analyzer
• Empower Chromatography Data Software
• MaxPeak High-Performance Surface (HPS) technology

Main results and discussion

Retention time reproducibility across three systems and nine conditions showed low intersystem standard deviations for 18 monitored peptides, confirming robust chromatographic performance. Overlay chromatograms under the most demanding conditions highlighted minimal shifts and consistent peak profiles. MaxPeak HPS technology reduced tailing factors of acidic peptides by up to 20 %, improving resolution and quantitation reliability. Automated data processing in Empower provided a single, “universal” processing method for both UV and MS data, with custom reports that rapidly display stress-induced attribute changes and peptide-level ratio thresholds.

Benefits and practical applications

The integrated LC-UV/MS workflow delivers:

• High reproducibility and confidence in peptide retention times across labs
• Improved peak shape for challenging analytes using MaxPeak HPS
• Streamlined, automated data analysis and reporting for rapid decision making
• Seamless transfer between upstream development and manufacturing QC environments

Future trends and opportunities

Advances in high-throughput column technologies, enhanced surface chemistries and AI-driven data processing are expected to further accelerate peptide mapping. Integration with multi-attribute methods and real-time monitoring platforms will enable deeper process understanding and automation in continuous bioprocessing. Development of libraries for universal processing templates across instrument vendors will simplify cross-laboratory standardization.

Conclusion

The ACQUITY Premier LC-UV/MS platform combined with Empower software and MaxPeak HPS provides a highly reproducible, automated peptide mapping workflow. This solution supports both process development and manufacturing QC, offering robust performance, improved peak quality and streamlined data analysis.

Reference

1. DeLaney K, Birdsall RE, Yu YQ. High Precision Performance of the ACQUITY Premier System (BSM Configuration) for Reproducible Peptide Mapping. Waters Application Note, April 2022; 720007593.
2. DeLaney K, Birdsall RE, Yu YQ. Utilizing Empower Software to Streamline and Automate the Detection of Sample-to-Sample Differences within Peptide Maps of Biopharmaceuticals. Waters Application Note, May 2022; 720007610.