Evaluating the Performance of Reversed-phase C18 Columns for Peptide Mapping of Monoclonal Antibodies

See how the Thermo Scientific™ Hypersil GOLD™ Peptide reversed-phase C18 column performs for peptide mapping, including hydrophilic peptide retention, PENNYK deamidation separation, and lot-to-lot consistency

Peptide mapping, using reversed-phase LC-MS workflows, supports biotherapeutic characterization by confirming primary sequence and assessing post-translational modifications (PTMs) such as deamidation and oxidation.

This application note evaluates the Thermo Scientific™ Hypersil GOLD™ Peptide reversed-phase C18 column for peptide mapping and compares it with cross-vendor C18 columns of the same format (2.1 × 150 mm), then examines lot-to-lot consistency across three column lots.

What’s You’ll Learn

Peptide mapping: Gain insight into CQAs and PTM monitoring, including deamidation and other challenges

Experimental design: Learn how to execute NISTmAb + rituximab digests, cross-vendor comparison, and lot-to-lot analysis

LC-MS conditions and data analysis workflow: Explore methods leveraging the Vanquish UHPLC, Orbitrap Exploris 240, BioPharma Finder, and Chromeleon data system

Performance readouts: Interpret sequence coverage data, peak area/RT reproducibility, peak asymmetry, mass accuracy, resolution, hydrophilic retention, carry-over, PTM quantitation

Lot-to-lot comparison: Evaluate three column lots for hydrophilic peptides, carry-over, and PTM abundance metrics

Who should download this?

• Biopharma analytical development and QC teams running peptide mapping or MAM-style LC-MS workflows
• Scientists comparing C18 column performance for peptide mapping (retention, resolution, repeatability)
• Labs troubleshooting early-eluting peptides (e.g., VSNK-region monitoring) or PTM separations (e.g., deamidation hot spots)

Download the application note to learn more