Comparison of alternative approaches to trypsin protein digestion for reproducible and efficient peptide mapping analysis of monoclonal antibodies

Importance of the Topic

Monoclonal antibodies represent the leading class of biotherapeutics due to high target specificity and favorable pharmacokinetics. Peptide mapping by tryptic digestion and LC-MS is regarded as the gold-standard for assessing critical quality attributes including sequence integrity and post-translational modifications. Reliable, high-throughput and reproducible sample preparation methods are essential for consistent characterization and quality control of antibody therapeutics.

Objectives and Study Overview

This work compares four approaches to trypsin digestion for peptide mapping of two model mAbs (adalimumab and NISTmAb RM 8671). The methods evaluated include traditional in-solution heat denaturation, a rapid guanidine-based protocol, the Thermo Scientific SMART Digest kit, and the Magnetic SMART Digest kit on a KingFisher Duo Prime system. Key metrics are digestion time, sequence coverage, reproducibility, and sample preparation–induced modifications such as deamidation and oxidation.

Methodology and Instrumentation

Samples were prepared in triplicate by different analysts across multiple days. Protocols varied in denaturation conditions (heat or chaotrope), digestion time (30 minutes to overnight), and pH. SMART Digest workflows employed thermally stable immobilized trypsin at 70 °C for 45 minutes, with or without magnetic bead support and full automation. LC separation used a Vanquish Flex Binary UHPLC with C18 column, and detection was performed on a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer. Data analysis utilized Thermo Scientific BioPharma Finder software for peptide identification and PTM quantification.

Main Results and Discussion

All methods achieved nearly 100 % sequence coverage of heavy and light chains in under 45 minutes for SMART workflows and under 16 hours for in-solution digestion. Chromatographic overlays revealed superior retention time reproducibility for the SMART Digest and magnetic workflows. In-solution heat protocols induced elevated levels of deamidation (up to 16 % at susceptible Asn sites) and oxidation (up to 11 % at Met residues). In contrast, SMART Digest methods at neutral pH limited deamidation to below 6 % and oxidation to below 4 %, with low standard deviations across replicates. Glycosylation profiles were consistent across methods, with major Fc glycans (A2G0F, A2G1F, A2G2F, high-mannose) quantified reproducibly.

Benefits and Practical Applications

• Rapid sample preparation in under 1 hour reduces turnaround time.
• Automatable workflows minimize manual handling and increase throughput.
• High sequence coverage with low preparation-induced artifacts improves data confidence.
• Robust digestion protocols support routine QC in biopharmaceutical environments.

Future Trends and Potential Applications

Integration of immobilized enzyme kits with advanced automation platforms will further enhance throughput and reproducibility. Extension of these workflows to other proteases and multi-attribute methods may enable deeper characterization of higher order structure and glycoform distributions. Coupling with AI-driven data analysis could accelerate decision making during process development and release testing.

Conclusion

SMART Digest and Magnetic SMART Digest approaches provide rapid, reproducible and fully automatable peptide mapping for monoclonal antibodies. They achieve complete sequence coverage while minimizing sample preparation-induced modifications, offering clear advantages over conventional in-solution protocols for biopharmaceutical QC.

Reference

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