Optimized digestion procedure and characterization for monoclonal antibodies and proteins by CESI-MS

Significance of the Topic

Peptide mapping by LC-MS remains a cornerstone in the characterization of monoclonal antibodies and biotherapeutic proteins. However, traditional reversed-phase approaches can struggle with extremes of peptide hydrophobicity, sample loss, and incomplete coverage of post-translational modifications. Sheathless capillary electrophoresis–mass spectrometry (CESI-MS) offers an orthogonal separation mechanism based on charge-to-mass ratio, delivering enhanced sensitivity, reduced ion suppression at nano-flow rates, and single-run analysis of diverse peptide classes.

Study Objectives and Overview

This work presents an optimized digestion procedure tailored for CESI-MS applications and its integration with the CESI 8000 Plus and TripleTOF 6600+ LC-MS/MS system. The protocol aims to achieve high sequence coverage of monoclonal antibodies, robust detection of glycopeptides, and reliable identification of low-level post-translational modifications in a single analysis.

Methodology and Instrumentation

• Sample Preparation: Antibody samples (e.g., adalimumab) were reduced with DTT, alkylated with iodoacetamide, and digested using trypsin in the presence of RapiGest SF surfactant. Digests were acid-quenched and diluted into a leading electrolyte (200 mM ammonium acetate, pH 4).
• Preconcentration and Separation: Transient isotachophoresis (t-ITP) followed by capillary zone electrophoresis on an OptiMS BFS silica capillary at ~40 nL/min enabled high-sensitivity loading and separation of peptides and glycopeptides.
• Mass Spectrometry: The TripleTOF 6600+ with NanoSpray Ion Source III and CESI adapter operated in positive mode using data-dependent acquisition. MS1 scans covered m/z 350–2250 with 150 ms accumulation, followed by up to 10 MS/MS scans per cycle at 50 ms each.

Main Results and Discussion

The optimized workflow delivered >99 % sequence coverage of adalimumab heavy and light chains from ~40 ng of digest. CESI-MS effectively resolved high-mannose glycopeptides and low-abundance species in a single run. Deamidation and oxidation sites were quantified, revealing 2–3 % deamidation on specific asparagine residues and 8.6 % methionine oxidation.

Benefits and Practical Applications

• Orthogonal separation enhances detection of hydrophobic and hydrophilic peptides often missed by LC-MS.
• Nano-flow CESI reduces ion suppression, improving sensitivity for low-level PTMs.
• Single-injection analysis streamlines quality control workflows in biopharmaceutical development and comparability studies.

Future Trends and Opportunities

Integration of CESI-MS with advanced data analysis and machine learning will further refine PTM mapping and biosimilarity assessment. Expanding the protocol to alternative proteases and multiplexed assays can accelerate comprehensive characterization of complex biologics in pharmaceutical QA/QC and research settings.

Conclusion

The combination of an optimized digestion protocol with the CESI 8000 Plus and TripleTOF 6600+ provides a powerful platform for high-coverage peptide mapping, enabling detailed characterization of monoclonal antibodies and their critical quality attributes in a single, sensitive analysis.

Reference

1. Improving the detection limits for highly basic neuropeptides using CESI-MS. SCIEX Technical Note, 2020.
2. Comprehensive analysis of low abundant mannose glycopeptides in peptide mapping of adalimumab. SCIEX Technical Note, 2020.
3. Gahoual R, Biacchi M, Chicher J, et al. Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry. MAbs. 2014;6(6):1464-1473.
4. Gahoual R, Burr A, Busnel JM, et al. Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry. MAbs. 2013;5(3):479-490.
5. Yu YQ, et al. A complete peptide mapping of membrane proteins: a novel surfactant aiding the enzymatic digestion of bacteriorhodopsin. Rapid Commun Mass Spectrom. 2004;18:711-715.
6. SCIEX. CESI 8000 High Performance Separation System User Manual, RUO-IDV-05-3897-C, 2020.
7. Lew C, Gallegos-Perez JL, Fonslow B, Lies M, Guttman A. Rapid level-3 characterization of therapeutic antibodies by capillary electrophoresis electrospray ionization mass spectrometry. J Chromatogr Sci. 2015;53(3):443-449.
8. Gahoual R, Busnel JM, Beck A, François YN, Leize-Wagner E. Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry. Anal Chem. 2014;86(18):9074-9081.