In-depth characterization of monoclonal antibodies

Importance of the topic

Monoclonal antibodies (mAbs) represent a major class of biotherapeutics used in oncology, immunology and other disease areas. Their structural heterogeneity, introduced through post-translational modifications (PTMs) such as glycosylation, oxidation and deamidation, can impact safety, efficacy and stability. Accurate intact mass measurement and detailed sequence mapping of mAbs and their subunits are therefore critical for biopharmaceutical development, quality control and regulatory compliance.

Objectives and study overview

This study evaluates the analytical performance of the Thermo Scientific™ Orbitrap™ Ascend BioPharma Tribrid™ mass spectrometer coupled to Vanquish™ Flex UHPLC for comprehensive characterization of trastuzumab. Two complementary workflows—intact mass analysis under native and denaturing conditions and middle-down mass spectrometry (MD-MS) of subunits—are developed to assess mass accuracy, sensitivity and sequence coverage.

Methodology and instrumentation

Sample preparation combines minimal handling with targeted digestion and reduction:

• Intact mAb: direct analysis of trastuzumab under native buffer (50 mM ammonium acetate) or denaturing conditions (0.1% formic acid, organic solvent).
• Subunit generation: Dithiothreitol (DTT) reduction to yield LC and HC subunits; IdeS protease digestion followed by DTT reduction to produce Fc/2, light chain (LC) and Fd′ subunits.

Liquid chromatography and mass spectrometry:

• Vanquish Flex UHPLC with MabPac SEC-1 size exclusion and MabPac RP columns for intact and subunit separations.
• Orbitrap Ascend BioPharma Tribrid MS operated in high-resolution intact protein mode (native/denaturing pressures) and low-pressure mode for subunits.
• Fragmentation techniques vary by application: electron transfer dissociation (ETD), electron transfer higher-energy dissociation (EThcD), ultraviolet photodissociation (UVPD) and proton transfer charge reduction (PTCR) for decongestion of product ions.

Instrumentation

Major instruments and consumables:

• Thermo Scientific Vanquish Flex UHPLC (system base, binary pump, autosampler, column compartment).
• Thermo Scientific MAbPac SEC-1 (5 µm, 4×150 mm) and MAbPac RP (4 µm, 2.1×50 mm) columns.
• Orbitrap Ascend BioPharma Tribrid mass spectrometer with Native MS, ETD, ETciD, EThcD, UVPD and PTCR capabilities.
• Reagents: Ammonium acetate, formic acid, acetonitrile, guanidine-HCl, DTT, Promega IdeS protease.

Main results and discussion

Intact mass analysis:

• Native vs. denaturing: trastuzumab retains higher charge states and m/z distribution under denaturing conditions; both conditions deliver baseline separation of common glycoforms.
• Sensitivity: intact mass profiles with accurate glycoform resolution down to 10 ng on column with mass errors within ±11 ppm.

Subunit mass analysis:

• Accurate mass measurements (<3 ppm) for LC, HC and Fc/2, Fd′ subunits using Xtract and ReSpect deconvolution.
• High-resolution settings (240 k) allow isotopic resolution for smaller subunits; lower settings (7.5 k) suffice for larger ones.

Middle-down fragmentation:

• ETD yields up to 80% sequence coverage for Fc/2, 75% for LC and Fd′; EThcD increases coverage to >90% for Fc/2 and reaches 91% for LC and 87% for Fd′.
• UVPD provides complementary a/x, b/y and c/z ions, raising coverage to >93% for 20–25 kDa subunits.
• PTCR reduces spectral congestion, further elevating sequence coverage for Fc/2 from 72% to 80% (EThcD) and from 66% to 75% (UVPD); combined EThcD, UVPD and PTCR across replicates achieves >90% coverage for Fc/2, LC and Fd′, and ~70% for the 50 kDa HC subunit.

Benefits and practical applications of the method

The integrated UHPLC–Orbitrap Ascend BioPharma platform delivers:

• Unmatched mass accuracy and resolution for intact proteins and glycoforms.
• Sensitive detection down to low nanogram levels, supporting limited sample availability.
• Versatile multi-technique fragmentation enabling in-depth sequence coverage.
• Rapid and robust workflows for routine QC, comparability and epitope mapping studies.

Future trends and applications

Advances in biopharma MS may include:

• Automation of middle-down workflows and data analysis pipelines with AI-driven deconvolution.
• Integration of native MS and ion mobility for conformer profiling of mAbs.
• Extension to next-generation biologics such as bispecifics, antibody–drug conjugates and glycoengineered variants.
• High-throughput screening in process development and lot release with real-time monitoring.

Conclusion

This study demonstrates that the Orbitrap Ascend BioPharma Tribrid mass spectrometer combined with Vanquish Flex UHPLC allows accurate intact mass profiling and comprehensive middle-down characterization of mAb subunits. The multi-technique fragmentation and PTCR capabilities achieve near-complete sequence coverage and high sensitivity, establishing a robust platform for biopharmaceutical analysis.

Reference

1. Srzentic K, Damoc E, Scheffler K et al. Complete characterization of monoclonal antibodies under native and denaturing conditions. Thermo Fisher Scientific Application Note, 2021.
2. Srzentic K, Damoc E. Full characterization of heterogeneous antibody samples under denaturing and native conditions on the Q Exactive BioPharma mass spectrometer. Thermo Fisher Scientific Application Note, 2017.
3. Kline JT, Mullen C, Durbin KR et al. Sequential ion–ion reactions for enhanced gas-phase sequencing of large intact proteins in a Tribrid Orbitrap mass spectrometer. J Am Soc Mass Spectrom. 2021;32(9):2334–2345.
4. Huguet R, Mullen C, Srzentić K et al. Proton transfer charge reduction enables high‐throughput top‐down analysis of large proteoforms. Anal Chem. 2019;91(24):15732–15739.
5. Scheffler K, Damoc E, Bailey A, Josephs J. Enabling mass spectrometric analysis of intact proteins in native conditions of a hybrid quadrupole-Orbitrap mass spectrometer. Thermo Fisher Scientific Poster, ASMS 2016.