Quick screening of intact antibody and antibody-drug conjugates with integrated microfluidic capillary electrophoresis and mass spectrometry

Importance of the Topic

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are cornerstone biotherapeutics in modern medicine. Rapid detection of molecular heterogeneity during development and manufacturing is critical to ensure drug safety, efficacy, and consistent quality. An integrated microfluidic capillary electrophoresis–mass spectrometry (CE-MS) platform offers a fast, sensitive, and low-consumption solution for intact protein profiling and variant quantification.

Objectives and Study Overview

This application note describes a workflow combining the ZipChip™ microfluidic CE device with a Thermo Scientific™ Q Exactive™ Orbitrap™ mass spectrometer and BioPharma Finder™ software to:

• Rapidly separate intact NIST reference mAb and a site-specific ADC analogue.
• Detect and quantify charge and glycoform variants within minutes.
• Demonstrate the dynamic range and mass accuracy of the platform for biotherapeutic screening.

Methodology

Sample Preparation:

• NIST mAb reference material and an engineered ADC bearing two DIBO-Biotin payloads were diluted to 0.5 µg/µL in water.

Capillary Electrophoresis:

• ZipChip HR chip (22 cm channel) with built-in ESI emitter.
• Background electrolyte: 10% isopropanol, 0.2% acetic acid.
• Pressure injection (2 psi, 5 s; ~0.65 nL) and separation under 770 V/cm with 2 psi assistance.
• Analysis time: ~3 minutes per run.

Data Processing:

• BioPharma Finder™ 2.0 software with sliding-window deconvolution (ReSpect™ algorithm).
• Protein sequence manager with specified fixed and variable modifications for mAb and ADC.

Instrumentation Used

• 908 Devices ZipChip™ microfluidic CE system (P/N 00950-01-00492) with HR chip (P/N 00950-01-00499).
• Thermo Scientific™ Q Exactive™ Plus Orbitrap™ MS with BioPharma option (P/N 0726055) in HMR mode (m/z 2500–6000).
• Thermo Scientific™ BioPharma Finder™ 2.0 software (P/N OPTON-30592).

Key Results and Discussion

mAb Analysis:

• Baseline separation of 0-, 1-, and 2-lysine variants within 3 minutes.
• Detection of five major glycoforms per lysine variant (G0F/G0F to G2F/G2F).
• Identification of 17 distinct variants with mass errors <20 ppm and relative abundances spanning over three orders of magnitude.

ADC Analysis:

• Near-baseline separation of lysine variants for the site-specific ADC.
• Clear mass spectra showing two DIBO-Biotin payload attachments (DAR 2) and low-abundance hexose adducts.
• Calculated DAR = 2.0 in agreement with the known payload.

Benefits and Practical Applications

• Fast throughput: <3 min per sample enables high screening capacity.
• Minimal sample consumption: ~0.2 ng per analysis.
• Online desalting and direct injection eliminate extensive sample prep.
• High-resolution separation reduces spectral complexity and enhances variant quantification.

Future Trends and Applications

Emerging directions for intact protein CE-MS include:

• Automation and higher-throughput microfluidic devices to support large screening campaigns.
• Integration of tandem MS for localization of post-translational modifications.
• Advanced data analytics and AI-driven deconvolution to accelerate variant identification.
• Extension to other biotherapeutic formats such as bispecific antibodies and fusion proteins.

Conclusion

The integrated ZipChip CE and Q Exactive Orbitrap MS workflow offers rapid, sensitive, and accurate profiling of intact mAbs and ADCs. Its ability to separate charge variants and detect minor glycoforms in under 3 minutes with low sample use makes it a powerful tool for biopharmaceutical development and quality control.

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