Selecting a Reversed-Phase Column for the Peptide Mapping Analysis of a Biotherapeutic Protein

Significance of the Topic

The peptide mapping of biotherapeutic proteins is a cornerstone technique in both proteomics research and quality control of biologic drugs. It provides detailed information on amino acid sequence coverage, post-translational modifications, and degradation products, ensuring product consistency and safety.

Objectives and Study Overview

This work compared ten different reversed-phase liquid chromatography columns to determine their suitability for peptide mapping using either 0.1% trifluoroacetic acid (TFA) or 0.1% formic acid (FA) ion-pairing mobile phases. A peptide standard mixture (MassPREP) and a tryptic digest of the NIST monoclonal antibody reference material (NISTmAb) served as test samples. Key performance metrics included peak capacity, retention of hydrophilic peptides, and selectivity differences among phases.

Methodology and Instrumentation

Samples: MassPREP Peptide Mixture (~15 µg/mL) and reduced/alkylated tryptic digest of NISTmAb (~0.1 mg/mL).
LC Conditions: ACQUITY UPLC H-Class Bio system, column temperature 60 °C, flow rate 0.2 mL/min, detection at 214 nm (UV) and ESI-MS (Xevo G2 Q-Tof).
Columns Evaluated: Waters Peptide BEH C18 (130 Å/300 Å, 1.7 µm), Peptide CSH C18 (130 Å, 1.7 µm & 2.5 µm), Peptide HSS T3 (100 Å, 1.8 µm), CSH Phenyl-Hexyl (130 Å, 1.7 µm), CORTECS C18 and C18+ (90 Å, 1.6 µm & 2.7 µm), CORTECS T3 (120 Å, 1.6 µm).

Main Results and Discussion

• Peak Capacity: Charged surface hybrid (CSH) C18 columns delivered the highest average peak capacities in both TFA and FA systems, outperforming standard BEH C18 phases by up to 20% in FA.
• Surface Charge: The positive charge on CSH particles improved peptide loading and peak narrowing, particularly in FA mobile phases.
• Particle Size: Decreasing particle size from 2.5 µm to 1.7 µm in CSH phases increased peak capacity by ~11–16%, while solid-core CORTECS C18+ showed a 15–17% gain going from 2.7 µm to 1.6 µm.
• Pore Size: Larger 300 Å pores benefited peptides above ~1.8 kDa by reducing restricted diffusion, offering modestly higher peak capacities than 130 Å phases.
• Hydrophilic Peptide Retention: HSS T3 (100 Å) demonstrated the greatest retention of small, hydrophilic peptides (e.g., RASG-1) under both TFA and FA conditions.
• Selectivity: Phenyl-hexyl chemistry reversed elution order of aromatic-rich peptides due to π–π interactions, while surface charge and base particle composition provided subtle but exploitable selectivity differences.

Benefits and Practical Applications

This comparative evaluation guides analysts in selecting columns that maximize resolution of critical quality attributes, ensure high sequence coverage (>95%), and adapt to either MS-friendly FA or high-performance TFA conditions. The recommended phases support both routine UV-based assays and LC-MS workflows in biopharmaceutical development, proteomics, and peptide purity testing.

Future Trends and Opportunities

Continued advancements may include tailored surface chemistries to target specific peptide classes, monolithic and sub-2 µm columns for ultra-high throughput, and machine-learning tools to predict optimal column selection. Integration with automated sample preparation and high-resolution mass spectrometry will further accelerate therapeutic protein characterization.

Conclusion

No single column suits all peptide mapping needs. However, a combination of charged surface hybrid and bridged-hybrid BEH C18 phases with varied pore and particle sizes offers a powerful starting point. Screening these columns under both TFA and FA conditions delivers robust, high-capacity separations with tunable retention and selectivity for comprehensive biotherapeutic protein analysis.

References

• Sandra K. et al. J. Chromatogr. A. 2014;1335:81–103.
• Sinha S. et al. Protein Sci. 2009;18:1573–1584.
• Neue UD. J. Chromatogr. A. 2005;1079:153–161.
• Wyndham KD. et al. Anal. Chem. 2003;75:6781–6788.
• Wang X. et al. Anal. Chem. 2006;78:3406–3416.
• Lauber MA. et al. Anal. Chem. 2013;85:6936–6944.
• Neue UD. et al. J. Chromatogr. A. 2006;1127:161–174.
• Krokhin OV, Spicer V. Anal. Chem. 2009;81:9522–9530.