An automated high-throughput workflow for peptide mapping to monitor post-translational modifications (PTMs) of monoclonal antibodies

Importance of the topic

Monoclonal antibodies represent a dominant class of biotherapeutics characterized by high molecular weight (~150 kDa) and structural complexity. Post-translational modifications (PTMs) such as glycosylation, deamidation, oxidation, glycation, N-terminal pyroglutamination and C-terminal lysine clipping introduce critical heterogeneity that can impact safety, efficacy and stability of these drugs.

Goals and overview of the study

This study establishes an automated, high-throughput peptide mapping workflow for comprehensive profiling of PTMs in five top-selling monoclonal antibodies (bevacizumab, cetuximab, adalimumab, rituximab and trastuzumab). Key objectives include evaluation of digestion reproducibility, peptide sequence coverage and confident PTM identification using magnetic bead-based tryptic digestion combined with high-resolution LC-MS/MS.

Methodology and instrumentation

• Sample preparation: Automated trypsin digestion using Thermo Scientific SMART Digest Bulk Magnetic Resin Kit on the KingFisher Duo Prime system (45 min at 70 °C, 96-well format).
• Chromatography: Thermo Scientific Vanquish Flex Binary UHPLC with Acclaim C18 column (2.2 μm, 2.1×250 mm), 65 min gradient, 0.3 mL/min.
• Mass spectrometry: Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap with HESI II source, data-dependent Top-5 MS/MS.
• Data analysis: Thermo Scientific BioPharma Finder 3.0 for automated peptide identification, sequence coverage mapping and PTM assignment.

Main results and discussion

• Retention time precision: ≤0.14% RSD (n=9) across technical replicates for cetuximab.
• Sequence coverage: 100% heavy and light chain coverage achieved for all five mAbs.
• PTM profiling:
  
  • Deamidation: Detected at multiple Asn/Gln sites (up to ~7% relative abundance).
  • Oxidation: Methionine/tryptophan oxidation levels <4%.
  • Glycosylation: Fc N-glycan variants (A2G0F, A2G1F, A2G2F, M5) quantified with low variability.
  • Glycation: Lysine glycation variants typically <1.6% abundance.
  • N-terminal pyroglutamination: Cyclization >85% in some mAbs.
  • C-terminal Lys loss: Clipping variants ranging ~67–97%.

Benefits and practical applications of the method

• Rapid, simplified digestion and sample handling (<2 h) with minimal hands-on time.
• Automated magnetic bead-based workflow increases throughput, reproducibility and consistency.
• High-confidence PTM profiling supports lot-to-lot comparability, stability studies and QC release testing.
• Integration with high-resolution UHPLC-MS/MS enables sensitive detection of low-abundance modifications.

Future trends and potential applications

• Application to other biotherapeutics such as fusion proteins and antibody-drug conjugates.
• Integration of informatics, machine learning and AI for advanced PTM pattern recognition.
• Adaptation to continuous manufacturing and in-line process monitoring.
• Development of multiplexed, comparative glyco- and peptido-typing workflows.

Conclusion

The automated magnetic bead-based SMART Digest protocol on the KingFisher Duo Prime, paired with Vanquish Flex UHPLC and Q Exactive Plus MS, provides a robust, efficient and reproducible workflow for complete peptide mapping of monoclonal antibodies. It delivers full sequence coverage, excellent retention time precision and reliable quantitation of critical PTMs, facilitating streamlined quality control and bioprocess development.

References

1. Reichert JM. Antibodies to watch 2017. MAbs. 2017;9(2):167–171.
2. Kaplon H, Reichert JM. Antibodies to watch in 2018. MAbs. 2018;10(2):183–203.
3. Huggett B. Public Biotech 2012–the numbers. Nat Biotechnol. 2013;31(8):697–703.
4. Liu H, Gaza-Bulseco G, Chumsae C. Glutamine deamidation of a recombinant monoclonal antibody. Rapid Commun Mass Spectrom. 2008;22(24):4081–4088.
5. Chelius D, Jing K, Lueras A, et al. Formation of pyroglutamic acid from N-terminal glutamic acid in antibodies. Anal Chem. 2006;78(7):2370–2376.
6. Higel F, Seidl A, Sorgel F, Friess W. N-glycosylation heterogeneity of monoclonal antibodies. Eur J Pharm Biopharm. 2016;100:94–100.
7. Wiegandt A, Meyer B. Characterization of N-glycans of cetuximab by LC-MS/MS and NMR. Anal Chem. 2014;86(10):4807–4814.
8. Haberger M, Heidenreich AK, Schlothauer T, et al. Functional assessment of antibody oxidation by native mass spectrometry. MAbs. 2015;7(6):1023–1030.
9. Chelius D, et al. Stability considerations for biopharmaceuticals: overview of degradation pathways. BioProcess Int. 2011;9(2):14–26.
10. Thermo Fisher Scientific. AN21835: Automated high-throughput workflow for peptide mapping of mAbs. 2018.