Sensitive profiling of IgG1 monoclonal antibody variants under native conditions

Significance of the Topic

Native mass spectrometry (MS) of intact monoclonal antibodies (mAbs) under non‐denaturing conditions has emerged as a rapid and informative approach for biopharmaceutical analysis. By preserving higher‐order structure and endogenous post‐translational modifications (PTMs), native MS enables clear separation of charge states and detailed characterization of proteoforms without extensive sample preparation. This capability is critical for ensuring product quality, monitoring microheterogeneity, and supporting decision making during early development and clone selection stages.

Aim and Study Overview

The primary goal of this study was to evaluate the performance of the Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer for high‐resolution accurate‐mass (HRAM) analysis of IgG1 mAb variants under native conditions. Two commercially available therapeutic mAbs (trastuzumab and a second IgG1) were profiled using size‐exclusion chromatography (SEC) and pH‐gradient cation‐exchange chromatography (CEX) coupled to native MS. Sensitivity, dynamic range, and the ability to detect low‐abundance glycoforms and charge variants were assessed.

Methodology

Chromatographic separations were performed under native conditions with volatile buffers:

• SEC: MAbPac™ SEC‐1 column (4.0 × 150 mm), isocratic 50 mM ammonium acetate, 0.3 mL/min at 30 °C.
• CEX: MAbPac™ SCX‐10 RS column (2.1 × 50 mm), pH gradient from 5.3 to 10.9 using ammonium bicarbonate/acetic acid to ammonium hydroxide at 0.4 mL/min, 25 °C.

Sample loads ranged from 0.05 to 50 µg to determine limits of detection (LOD) and quantitation (LOQ).

Instrumentation Used

• LC System: Thermo Scientific™ Vanquish™ Duo UHPLC with dual binary pumps and split sampler for tandem LC‐MS operation.
• Mass Spectrometer: Orbitrap Exploris™ 240 with BioPharma Option, operating at 30,000 FWHM resolution at m/z 200 under optimized native ESI conditions.
• Software: Xcalibur™ 4.2 for data acquisition and Thermo Scientific™ BioPharma Finder™ 4.0 with Sliding Window and ReSpect™ algorithms for data processing.

Main Results and Discussion

SEC‐MS analysis of trastuzumab across a dilution series demonstrated:

• Clear detection of m/z 5,000–7,000 charge envelopes down to 0.05 µg on column.
• LOD of 0.03 µg and LOQ of 0.08 µg for intact mAb mass profiling.
• Reproducible high‐quality spectra and confident mass accuracy even at low sample loads.

CEX‐MS profiling of mAb #2 revealed:

• Separation of major and minor charge variants, including a triply deamidated acidic species at low abundance.
• Detection of species present at ≤3 % relative abundance due to exceptional sensitivity.

The wide dynamic range and spectral clarity enabled confident structural assignments and microheterogeneity assessment without denaturation.

Benefits and Practical Applications

This native MS workflow offers:

• Operational simplicity through standardized tune conditions transferable between instruments.
• Rapid, label‐free detection of glycoforms and charge variants at early development stages, where sample is scarce.
• Enhanced confidence in identity testing and PTM monitoring for QA/QC and biopharmaceutical characterization.

Future Trends and Opportunities

Advances in native MS are expected to include:

• Integration with orthogonal separation techniques (e.g., ion mobility) for deeper conformational analysis.
• High‐throughput automation and real‐time monitoring of PTMs during process development.
• Broader application to diverse biologics, including bispecific antibodies and complex multimers.
• Regulatory acceptance of HRAM native MS as a standard tool for product characterization.

Conclusion

The Orbitrap Exploris 240 demonstrates exceptional sensitivity, mass accuracy, and dynamic range for native MS analysis of intact mAbs. Coupling SEC and CEX with HRAM detection enables comprehensive profiling of glycoforms and charge variants from minimal sample loads, supporting accelerated biopharmaceutical development and robust quality control.

Reference

• 1. Tassi M. et al. Journal of Separation Science 2017, 41, 125–144.
• 2. Füssl F. et al. Analytical Chemistry 2018, 90(7), 4669–4676.
• 3. Füssl F. et al. mAbs 2018, 11(1), 116–128.