Characterization of monoclonal antibodies and antibody drug conjugates using iCIEF-MS online coupling platform

Importance of the Topic

Monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) are advanced biotherapeutics whose efficacy and safety critically depend on precise characterization of charge variants. Charge heterogeneity can arise from post-translational modifications, truncations or drug payloads, influencing potency, stability and immunogenicity. Integrating high-resolution separation with mass spectrometry accelerates variant profiling and supports rigorous quality control in biopharmaceutical development.

Objectives and Study Overview

This work aimed to establish and validate an online coupling of imaged capillary isoelectric focusing (iCIEF) with high-resolution accurate mass (HRAM) Orbitrap MS. The study focused on two targets:

• Pembrolizumab, a humanized anti-PD-1 mAb with multiple acidic and basic variants.
• T-DM1 (trastuzumab emtansine), an ADC carrying cytotoxic DM1 with a range of drug-to-antibody ratios (DAR D0–D10).

The goal was real-time intact analysis of charge variants, reduction of spectral complexity and detection of low-abundance species.

Methodology and Used Instrumentation

Samples were prepared at 1 mg/mL with appropriate ampholyte mixtures. Separation employed a CEInfinite iCIEF platform using an AES cartridge (ID 200 µm) under voltage gradients (1 000–3 000 V). Mobilization used acetic/formic acid buffers with ACN make-up flow.
Online detection was performed on a Thermo Scientific Q Exactive Plus BioPharma Orbitrap mass spectrometer (resolution 35 000, spray voltage 3.9 kV, capillary temp 275 °C). Data processing utilized BioPharma Finder software for deconvolution and PTM assignment.

Main Results and Discussion

• Pembrolizumab: Eight charge variants (four acidic, three basic, one main) were baseline resolved. MS deconvolution identified heavy chain N-terminal pyroglutamate conversion, C-terminal truncations (–1 K, –2 GK), oxidation and deamidation. Even trace variants (~0.3 %, ~4.8 ng) yielded high signal-to-noise ratios.
• T-DM1 ADC: iCIEF separated D0–D10 payload forms at the UV level, impossible by conventional RPLC-MS. Average DAR was calculated as 3.68. Online coupling significantly simplified MS spectra by isolating single drug-load species, reducing overlapping charge states and facilitating unambiguous mass assignment.

Benefits and Practical Applications

• Real-time intact mass profiling of mAbs and ADCs without offline fraction collection.
• Enhanced detection of low-level variants improves product characterization and comparability studies.
• Simplified MS spectra accelerate data interpretation and reduce analysis time.
• Supports quality control, stability testing and biosimilar evaluation in biomanufacturing.

Future Trends and Potential Applications

• Integration with automated data analysis pipelines for high-throughput screening.
• Expansion to next-generation modalities (bispecifics, fusion proteins, novel ADC linkers).
• Coupling with orthogonal techniques (peptide mapping, glycan profiling) to build comprehensive multi-attribute methods.
• Real-time in-process monitoring to support continuous manufacturing workflows.

Conclusion

The direct online coupling of iCIEF with Orbitrap MS provides a streamlined, high-resolution workflow for intact charge variant analysis of mAbs and ADCs. This approach delivers clear separation, simplified mass spectra and sensitive detection of minor species, enabling robust characterization for research and QC applications.

Reference

• Zhang X, Zhou Z, Sang M, Huang T, Chen T, Bo T, Du M. In Depth Characterization of mAb Charge Variants with iCIEF Fractionation Followed by Peptide Mapping Analysis. Thermo Fisher Scientific; 2021.