Consistent results for peptide mapping and monitoring across three systems of the Vanquish UHPLC platform

Importance of the Topic

Peptide mapping and monitoring by UHPLC coupled to high-resolution Orbitrap mass spectrometry is central to biopharmaceutical characterization and quality control. Reliable retention times, reproducible quantitation of post-translational modifications, and seamless method transfer across laboratories accelerate development of protein therapeutics and ensure regulatory compliance.

Goals and Study Overview

This study evaluated consistency, reproducibility, and method transferability of peptide mapping and monitoring across three Thermo Scientific Vanquish UHPLC systems (Horizon, Flex, Duo) using two protein digest samples (BSA and NISTmAb) run under 30 min and 90 min gradients. Each system performed ten replicate injections per sample on each column, with a second data acquisition two weeks later to assess intermediate precision.

Methodology and Instrumentation

• Sample preparation: BSA predigested by supplier; NISTmAb reduced, alkylated, buffer-exchanged, and trypsin-digested under controlled conditions.
• Chromatography: Two Acclaim Vanquish C18, 2.1 × 250 mm, 2.2 µm columns; 30 min (BSA) and 90 min (NISTmAb) gradients with reconditioning; method translation via Chromeleon CDS.
• UHPLC systems: Horizon (1,500 bar), Flex (binary pumps, 340 µL GDV), Duo (tandem LC-MS mode, two pump setup, 175 µL GDV per pump).
• Mass spectrometry: Q Exactive Plus Orbitrap; data-dependent MS/MS for mapping, full-MS for targeted monitoring.
• Data analysis: Peptide identification and PTM assessment with BioPharma Finder 4.1; targeted quantitation in Chromeleon CDS 7.3.

Main Results and Discussion

• Retention time precision was excellent (RT CV ≤ 2%) on all systems. The Flex system exhibited a consistent ~46 s shift under the 30 min gradient due to higher gradient delay volume; negligible shift under the 90 min gradient.
• Sequence coverage was identical across platforms (100 % heavy chain; 98.6 % light chain).
• Peak area CVs remained below 15 % for all peptides; PTM quantitation showed CVs mostly under 10 % even for modifications down to 0.5 % abundance.
• Comparative method transfer via automatic gradient translation in Chromeleon CDS preserved chromatographic performance and quantitation across instruments.

Benefits and Practical Applications

• Highly reproducible qualitative and quantitative peptide mapping and monitoring across multiple UHPLC systems.
• Seamless method transfer using Chromeleon CDS automatic translation reduces revalidation effort.
• Vanquish Duo in tandem LC-MS mode increases mass spectrometer usage efficiency, boosting throughput by 23 %–46 %.

Future Trends and Applications

Hardware modifications to harmonize gradient delay volumes and advanced automated workflows will further streamline multi-attribute method (MAM) implementation. Integration with machine learning-driven data processing promises faster, more consistent PTM profiling and higher throughput in biopharma QC and research.

Conclusion

All three Vanquish UHPLC systems delivered highly consistent retention times, peak shapes, and quantitative PTM measurements for peptide mapping and monitoring. Method transferability across Horizon, Flex, and Duo platforms was achieved with minimal adjustments, supporting robust MAM workflows and increased analytical throughput.

Instrumentation Used

• Thermo Scientific Vanquish Horizon, Flex Binary, and Duo UHPLC systems
• Thermo Scientific Q Exactive Plus Orbitrap mass spectrometer
• Thermo Scientific Acclaim Vanquish C18 column, 2.1 × 250 mm, 2.2 µm
• Thermo Scientific Chromeleon CDS 7.3 and BioPharma Finder 4.1 software

References

1. Overcoming the challenges of liquid chromatography method transfer: A CDMO perspective. Thermo Scientific Case Study 000565.
2. Method transfer onto the Thermo Scientific Vanquish HPLC/UHPLC platform: A CDMO perspective. Thermo Scientific Case Study 000566.
3. High-throughput peptide mapping of trastuzumab using a tandem LC-MS workflow. Thermo Scientific Application Note 73784.
4. Tandem UHPLC operation for high-throughput LC-MS peptide mapping analyses. Thermo Scientific Application Note 72203.