ATE: Capillary Electrophoresis – Mass Spectrometry for Intact Mass Analysis of Antibodies and Antibody-Drug-Conjugate

Significance of Capillary Electrophoresis–Mass Spectrometry for Antibody Analysis

Accurate characterization of monoclonal antibodies and antibody–drug conjugates is critical for biopharmaceutical quality control, lot release and early development. Charge heterogeneity and glycoform distribution affect safety, efficacy and stability. Combining high‐resolution capillary electrophoresis with Orbitrap mass spectrometry addresses challenges in top‐down analysis of intact biomolecules, offering rapid separation and precise mass measurement without extensive sample preparation.

Objectives and Study Overview

This work aimed to develop and demonstrate a streamlined workflow for intact mass analysis of NIST reference monoclonal antibody (mAb RM 8671) and its model antibody–drug conjugates (ADCs). Key goals included separation of charge variants, identification of glycoforms, and confirmation of payload conjugation using microfluidic CE coupled to a Thermo Scientific Q Exactive HF Biopharma mass spectrometer with Biopharma Finder software.

Methodology and Instrumentation

The NIST mAb standard was analyzed by high‐field microfluidic CE (ZipChip) with a 22 cm channel using 0.2% acetic acid/10% IPA (pH 3.17) as background electrolyte. Sample injection (0.2 ng protein) and separation under 20 kV completed in under 3 minutes. The CE outlet was interfaced to a Q Exactive HF Biopharma mass spectrometer operating in intact protein mode (m/z 2500–6000, 6 scans/sec, in‐source CID 100 eV). ADCs bearing AF488, biotin or Dylight800 payloads were prepared by click chemistry on N‐glycans. Data were processed with BioPharma Finder 2.0, using ReSpect deconvolution and reference database matching.

Main Results and Discussion

• Three charge variants of NIST mAb (0, 1 or 2 C‐terminal lysines) were baseline‐separated by CE based on mobility shifts from additional positive charges.
• At least 15 distinct glycoforms were identified across lysine variants, including major G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2F and G2F/G2F pairs, with relative abundances spanning three orders of magnitude.
• ADC conjugates exhibited the expected heterogeneity; AF488 and biotin ADCs showed exclusively di‐payload species, confirming high click chemistry yield. Dylight800 ADC displayed a more complex profile requiring further optimization.
• The mild acidic separation conditions preserved near‐native charge state distributions (mAb envelope 26–31), facilitating accurate mass assignment up to ~150 kDa.

Benefits and Practical Applications

This CE–MS approach enables rapid, high‐resolution profiling of intact mAbs and ADCs without desalting or extensive sample cleanup. It supports biopharmaceutical development by providing detailed proteoform maps, charge variant resolution and payload quantification in under 3 minutes per run.

Future Trends and Potential Applications

Future developments may include integration with automated sample handling, expansion to higher mass range instrumentation, and real‐time process monitoring. Enhanced software algorithms for deconvolution and database matching will further improve throughput and confidence in complex proteoform identification, extending applicability to diverse biotherapeutics.

Conclusion

The combination of microfluidic CE and high‐resolution Orbitrap MS coupled with dedicated software offers an efficient top‐down platform for intact antibody and ADC characterization. This method delivers rapid separation of charge and glycoforms, clear identification of conjugate species and minimal sample preparation, addressing critical needs in biopharmaceutical analytics.

Instrumentation

• 908 Devices ZipChip microfluidic CE platform
• Thermo Scientific Q Exactive HF Biopharma Orbitrap mass spectrometer
• BioPharma Finder 2.0 software with ReSpect deconvolution

References

1. Schiel J.E., Davis D.L., Borisov O.V. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization: The NISTmAb Case Study. ACS Publications, 2015.
2. Redman E.A., Mellors J.S., Starkey J.A., Ramsey M. Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis–Mass Spectrometry. Anal. Chem. 2016;88:2220–2226.