Complete characterization of monoclonal antibodies under native and denaturing conditions

Significance of the Topic

Therapeutic monoclonal antibodies exhibit complex glycosylation and a variety of post-translational modifications that impact their safety, efficacy and stability. Comprehensive mass spectrometric characterization under both native and denaturing conditions is essential to monitor proteoform heterogeneity, confirm structural integrity, and support biopharmaceutical development and quality control.

Objectives and Study Overview

• Evaluate the Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer with BioPharma Option for full antibody characterization.
• Establish workflows for intact mass analysis (native and denatured), subunit analysis (Top-Down/Middle-Down), and peptide mapping.
• Use trastuzumab as a model monoclonal antibody to demonstrate sequence coverage, proteoform identification, and PTM localization.

Methodology and Instrumentation

Samples were analyzed at three levels: intact protein under native and denaturing conditions, ~23–50 kDa subunits generated by chemical reduction or IdeS digestion plus reduction, and tryptic peptides using SMART Digest kits. Liquid chromatography employed size-exclusion (MAbPac SEC-1) for native, reversed-phase MAbPac RP for denaturing and subunits, and Acclaim VANQUISH C18 for peptides. Mass analysis was performed on an Orbitrap Exploris 480 with BioPharma Option using Intact Protein and Peptide application modes, adjustable IRM pressure, and preconfigured method templates. Data were processed in BioPharma Finder 4.0 using ReSpect, Xtract and Top-Down algorithms for deconvolution and sequence identification.

Key Results and Discussion

• Intact native analysis resolved glycoforms (+23 to +29 charge states) with mass errors ≤6.2 ppm; denaturing analysis detected +39 to +70 charge states with ≤4.8 ppm error.
• Reduced light and heavy chains (24 and 50 kDa) yielded isotopically resolved spectra; mass accuracies of 1.7–2.8 ppm were achieved using high and low resolution settings.
• Top-Down/Middle-Down fragmentation delivered 62% sequence coverage of the light chain and 36.5% of the heavy chain in a single experiment; IdeS-derived subunits achieved ~50% coverage each.
• Peptide mapping attained 100% sequence coverage for both chains and confidently identified methionine oxidation and other PTMs with high mass accuracy.

Benefits and Practical Applications

• Integrated LC-MS platform supports multiple characterization workflows without extensive method development.
• High sensitivity and mass accuracy enable detection of low-abundance proteoforms and PTMs.
• Adjustable IRM pressure improves analysis of large native complexes and isotopically resolved subunits on the same instrument.
• Predefined templates accelerate method transfer and assay reproducibility across development and QC labs.

Future Trends and Opportunities

Advances may include automated multi-attribute methods, real-time data processing with machine learning, deeper structural interrogation of glycoforms and higher-order complexes, and broader application to emerging modalities such as bispecific antibodies and antibody–drug conjugates.

Conclusion

The Orbitrap Exploris 480 with BioPharma Option delivers a versatile, high-performance solution for complete monoclonal antibody characterization. Its combined native/denaturing, Top-Down/Middle-Down, and peptide mapping capabilities provide detailed proteoform, sequence, and PTM insights critical for biotherapeutic development and quality assurance.

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