Development of a systematic approach for improving tryptic digestion, recovery and chromatographic performance for mAb analysis

Significance of the Topic

Peptide mapping by tryptic digestion and LC-MS/MS is a cornerstone analytical technique in biopharmaceutical development for confirming primary structure and monitoring critical post-translational modifications. Achieving consistent and complete proteolysis while ensuring robust chromatographic performance is essential for accurate identification and quantitation of protein attributes.

Objectives and Study Overview

This study aimed to develop a systematic, automated workflow that minimizes missed cleavages during digestion of monoclonal antibodies (mAbs) and improves chromatography of hydrophobic peptides. Three digestion protocols were compared: a modified two-step SMART Digest method, the original one-step SMART Digest, and a conventional in-solution MAM protocol. Chromatographic conditions on C4 versus C18 stationary phases were also evaluated.

Methodology and Instrumentation

The automated digests were performed on a KingFisher Duo Prime system using temperature-stable immobilized trypsin beads. Method 1 employed a two-step protocol (75 °C for 15 min, then 40 °C for 30 min) with fresh resin for each step. Method 2 used a single 75 °C, 30 min digestion. Method 3 was a conventional in-solution digest with reduction, alkylation, buffer exchange, and a 37 °C trypsin step.

• Chromatography: Thermo Vanquish UHPLC with Accucore and Hypersil GOLD C4/C18 columns.
• Mass spectrometry: Orbitrap Fusion Tribrid with HESI source, data-dependent acquisition.
• Sample cleanup: GuHCl wash steps integrated into the KingFisher program to recover hydrophobic peptides.

Main Results and Discussion

The two-step SMART Digest protocol yielded a marked decrease in missed cleavages: on average 60 % of peptides showed zero missed cleavages versus ~32–34 % for the alternative methods. This simplifies data analysis by consolidating modified residues into single peptide species. Comparing stationary phases, the C4 column substantially reduced peak tailing and carry-over of hydrophobic peptides compared to C18, without loss of sequence coverage. Early-eluting peptides detected between 1.3 and 3 min prompted reducing the waste diversion time to 1.2 min. Incorporating an 8 M guanidine-HCl wash of resin and comb surfaces prevented time-dependent adsorption of hydrophobic peptides and enhanced their recovery.

Benefits and Practical Applications

• Automated, robust digestion with lower variability and reduced manual intervention.
• Enhanced peptide map clarity, aiding precise quantitation of modifications.
• Improved chromatographic peak shapes and minimized carry-over on C4, streamlining high-throughput analysis.

Future Trends and Applications

Integration of high-throughput automated digestion workflows with advanced column chemistries and shorter gradients will further accelerate biopharmaceutical characterization. Emerging microflow-MS and machine learning–assisted data processing promise even greater sensitivity and speed. Continued optimization of sample preparation, including alternative proteases and novel surface coatings, may yield deeper coverage and higher reproducibility.

Conclusion

A modified two-step SMART Digest protocol combined with C4 chromatography and targeted wash steps offers a streamlined, reproducible approach for mAb peptide mapping. This workflow significantly reduces missed cleavages and chromatographic artifacts, supporting reliable structural and quality attribute analysis in biopharmaceutical development.

References

1. Jakes C et al. Tracking the Behavior of Monoclonal Antibody Product Quality Attributes Using a Multi-Attribute Method Workflow. J Am Soc Mass Spectrom 32:1998–2012 (2021).
2. Kristensen DB et al. Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides. J Anal Chem 94(49):17195–17204 (2022).