Analysis of Monoclonal Antibodies and Their Fragments by Size Exclusion Chromatography Coupled with an Orbitrap Mass Spectrometer

Importance of the Topic

Monoclonal antibodies (mAbs) have become cornerstone therapeutics in oncology, immunology and other disease areas. Regulatory agencies require detailed characterization of mAb purity, aggregation state, charge variants and glycosylation patterns to ensure product safety and efficacy. Size-exclusion chromatography (SEC) is routinely used for aggregate quantitation but lacks mass accuracy; coupling SEC with high-resolution mass spectrometry (SEC-MS) addresses this gap and supports comprehensive quality control of biotherapeutics.

Objectives and Study Overview

This study aimed to establish SEC-MS as a unified platform for both intact mAb and fragment analysis. By combining a volatile SEC buffer with an Orbitrap mass analyzer, the authors demonstrated native mass measurement of whole antibodies under near-physiological conditions and high-accuracy analysis of heavy chain (HC), light chain (LC), Fab and Fc fragments after reduction or enzymatic digestion.

Methodology and Instrumentation

Sample Preparation:

• Intact mAb analyzed directly under non-denaturing SEC conditions.
• Reduction: 20 mM DTT treatment at 50 °C to yield HC and LC subunits.
• Papain digestion: generation of Fab and Fc fragments in Tris-HCl buffer, pH 7.6.

Chromatography:

• Native SEC mobile phase: 20 mM ammonium formate, pH 6.3 (or 20 mM ammonium acetate, pH 6.8) to preserve quaternary structure.
• Denaturing SEC mobile phase: 20 % acetonitrile, 0.1 % formic acid, 0.05 % TFA for fragment separation.

Mass Spectrometry:

• Thermo Scientific Exactive Plus EMR Orbitrap, m/z range 350–20 000 in EMR mode for native analysis.
• Denaturing mode: 400–6000 m/z or 2000–4000 m/z, resolution up to 35 000, HCD = 10–100 eV, capillary temperature 275 °C, electrospray voltage 4.3 kV.
• Data deconvolution: Thermo Scientific Protein Deconvolution 2.0 with ReSpect algorithm.

Used Instrumentation

• Thermo Scientific Dionex UltiMate 3000 RSLCnano System with SRD-3400 degasser, NCS-3500RS pump, WPS-3000TPL autosampler.
• MAbPac SEC-1 column (5 µm, 4 × 300 mm).
• Thermo Scientific Exactive Plus EMR Orbitrap mass spectrometer.

Main Results and Discussion

Non-denaturing SEC-MS with ammonium formate produced intact mAb profiles featuring a shifted high m/z charge envelope (m/z ∼5100–6200) and deconvoluted mass ~148 033 Da. Adjacent peaks indicated glycoforms (+162 Da per hexose) and a C-terminal lysine variant (+129 Da). Ammonium acetate buffer caused peak broadening.

Under denaturing conditions, HC and LC were baseline-separated. The HC deconvoluted mass (~50 614 Da) showed glycoform and lysine variants; the LC (~23 403 Da) exhibited a single non-glycosylated form. Fab and Fc fragments eluted at ~9.9 min and ~10.8 min, with Fc masses around 52 753 Da (glyco/lysine variants) and Fab at ~47 318 Da, enabling rapid fragment-specific profiling.

Benefits and Practical Applications

SEC-MS offers a streamlined workflow for intact mass and fragment analysis without extensive desalting or low-pH denaturation. Native buffer compatibility preserves higher-order structure, while high-resolution MS ensures accurate mass assignment of PTMs. This approach accelerates QC testing for mAbs, antibody-drug conjugates and biosimilars.

Future Trends and Potential Applications

• Integration with automated glycan profiling and peptide mapping.
• Application to antibody-drug conjugate characterization and real-time bioprocess monitoring.
• Combining SEC-MS with ion mobility or higher-throughput UHPLC systems.
• Broader adoption in regulatory frameworks for biotherapeutic analysis.

Conclusion

The combination of volatile SEC mobile phases and Orbitrap MS provides a powerful platform for native intact mAb measurement and detailed fragment analysis. This methodology supports comprehensive characterization of antibody heterogeneity, glycosylation and variant forms in a single workflow suitable for research and QC environments.

Reference

1. Lin S., Wang H., Hao Z., Bennett P., Liu X. Analysis of Monoclonal Antibodies and Their Fragments by Size Exclusion Chromatography Coupled with an Orbitrap Mass Spectrometer. Thermo Fisher Scientific Poster Note PN20912_WCBP2014, 2014.