High-throughput peptide mapping of trastuzumab using a tandem LC-MS workflow

Significance of the Topic

The detailed mapping of peptide fragments from monoclonal antibodies is a cornerstone of biopharmaceutical quality control. High-throughput peptide mapping ensures rapid, accurate characterization of critical quality attributes such as post-translational modifications (PTMs). By minimizing instrument idle time and maximizing sample throughput, laboratories can maintain regulatory compliance, improve batch-to-batch consistency, and accelerate therapeutic development.

Objectives and Overview of the Study

This study evaluates a tandem LC-MS workflow based on the Thermo Scientific Vanquish Duo UHPLC system combined with an Orbitrap Exploris 240 mass spectrometer. The primary goals are:

• To demonstrate seamless method setup using Chromeleon CDS for dual-column operation.
• To compare analysis time and data quality against conventional single-column peptide mapping.
• To assess retention time reproducibility and PTM quantification across 21 alternating injections of trastuzumab digest.

Methodology

Sample preparation employed SMART Digest Trypsin kits with magnetic resin on a KingFisher Duo Prime platform. Tryptic digestion was fully automated, saving up to 20 hours per batch compared to in-solution protocols. Chromatographic separation used a 0.3 mL/min gradient of water and acetonitrile (both 0.1% formic acid) on Acclaim VANQUISH C18 columns (2.1×250 mm, 2.2 µm). The tandem workflow alternated analytical gradients and wash/reconditioning steps across two pumps and columns. Mass spectrometry was performed at 120 000 FWHM resolution on the Orbitrap Exploris 240 with BioPharma Option. Data acquisition and processing utilized Chromeleon CDS 7.2.10 and BioPharma Finder 4.0.

Used Instrumentation

• Vanquish Horizon Duo UHPLC System with dual Binary Pump H, Split Sampler HT, and Column Compartment H
• Acclaim VANQUISH C18 columns (2.1×250 mm, 2.2 µm)
• Orbitrap Exploris 240 mass spectrometer with BioPharma Option
• Chromeleon CDS software and BioPharma Finder

Main Results and Discussion

Chromatograms from both channels showed near-perfect overlap, with retention time RSDs below 1.5% across five representative peptides over 21 injections. PTM quantification for deamidation, oxidation, glycation, isomerization, and glycoforms yielded RSDs under 10% for abundant species. Low-level modifications exhibited RSDs up to 20%, still within acceptable QC limits. The tandem mode reduced mass spectrometer idle time by at least 6 hours in a 24 hour period, achieving over 25% faster throughput compared to standard LC operation.

Benefits and Practical Applications

• Significant reduction in total analysis time without loss of chromatographic resolution.
• High reproducibility of retention times and PTM measurements across two parallel channels.
• Automated sample digestion and standardized software templates support cGMP compliance and instrument-to-instrument method transfer.
• Enhanced laboratory productivity for routine QC, stability studies, and multi-attribute method (MAM) workflows.

Future Trends and Potential Applications

Integration of tandem LC-MS with advanced MAM data analysis and machine learning is expected to further streamline QC workflows. Miniaturized dual-column systems, inline sample preparation, and real-time process monitoring are emerging trends. Broader adoption of automated, high-throughput mapping will accelerate biosimilar development and support adaptive manufacturing in continuous bioprocessing.

Conclusion

The Vanquish Duo UHPLC system in tandem-mode with an Orbitrap Exploris 240 delivers robust, high-throughput peptide mapping for monoclonal antibodies. The workflow offers excellent reproducibility, significant time savings, and straightforward method setup in Chromeleon CDS. Combined with automated digestion, this platform enhances QC efficiency while maintaining or improving data quality.

References

1. FDA-CDER-CBER. Quality Considerations in Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product. Guidance for Industry. April 2015.
2. Rogstad S, Faustino A, Ruth A, Keire D, Boyne M, Park J. A Retrospective Evaluation of the Use of Mass Spectrometry in FDA Biologics License Applications. J Am Soc Mass Spectrom. 2017;28:786–94.
3. Rogers RS, Nightlinger NS, Livingston B, Campbell P, Bailey R, Balland A. Development of a Quantitative Mass Spectrometry Multi-Attribute Method for Characterization, Quality Control Testing, and Disposition of Biologics. MAbs. 2015;7:881–90.
4. Rogers RS, Abernathy M, Richardson DD, Rouse JC, Sperry JB, Swann P, et al. A View on the Importance of “Multi-Attribute Method” for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy. The APPS Journal. 2018;20:1–8.
5. Thermo Scientific. SMART Digest compared to classic in-solution digestion of rituximab for in-depth peptide mapping characterization. Application Note 72141.