Characterization of intact monoclonal antibody with microfluidic chip electrophoresis mass spectrometry

Importance of Topic

The structural microheterogeneity of therapeutic monoclonal antibodies is critical for their safety and efficacy in biopharmaceutical production.
Charge variants such as lysine clipping and deamidation, as well as diverse glycoforms, must be characterized rapidly and accurately.

Objectives and Study Overview

This study aimed to develop a fast intact protein method to separate and identify multiple proteoforms of the NIST reference monoclonal antibody RM 8671.
The approach couples microfluidic capillary electrophoresis with high-resolution mass spectrometry to resolve charge and glycosylation variants within minutes.

Methodology and Instrumentation

The NISTmAb was analyzed without desalting at 0.5 μg/μL in 0.2 % acetic acid/10 % IPA (pH 3.17).
Microfluidic CE separation was performed on a ZipChip device (22 cm channel) with 20 kV separation voltage and 2 kV nano-ESI.
Mass detection used a Thermo Scientific Q Exactive BioPharma Orbitrap mass spectrometer under mild denaturing conditions (m/z 2500–6000, 6 scans/sec, in-source CID 100 eV).
Data deconvolution and proteoform assignment employed Thermo’s BioPharma Finder software with the ReSpect algorithm.

Main Results and Discussion

Microfluidic CE-MS achieved baseline separation of 0, 1, and 2 C-terminal lysine variants of the intact mAb in under four minutes.
Five dominant glycoforms (G0F/G0F, G0F/G1F, G1F/G1F, G1F/G2F, and G2F/G2F) were identified for each lysine state, along with minor 1–2 lysine adducted glycoforms.
Deconvoluted masses matched theoretical values within <10 ppm for major forms and up to ~29 ppm for low-abundance species.
The method delivered three orders of magnitude dynamic range and captured >90 % of known NISTmAb proteoforms.

Benefits and Practical Applications of the Method

Ultrafast intact mAb profiling reduces analysis time and simplifies sample preparation by eliminating desalting steps.
The high resolution of microfluidic CE and Orbitrap MS enables simultaneous charge and glycoform mapping in a single run.
This workflow supports quality control and comparability studies in biopharma, facilitating batch release and biosimilar assessment.

Future Trends and Potential Applications

Integration of microfluidic CE-MS with automation and bioinformatics will further accelerate biotherapeutic characterization.
Advances in higher-throughput CE chips and real-time data processing may extend this approach to multispecific antibodies and ADCs.
Combining intact and subunit analyses in a unified platform could enhance structural insight into complex protein modalities.

Conclusion

The combination of high-field microfluidic CE separation and high-resolution Orbitrap MS provides a rapid, robust platform for intact mAb heterogeneity analysis.
This method resolves C-terminal lysine variants and glycoforms within minutes, achieving comprehensive proteoform coverage and supporting critical biopharmaceutical workflows.

Reference

• Schiel JE, Davis DL, Borisov OV. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization: The NISTmAb Case Study. ACS 2015.
• Redman EA, Mellors JS, Starkey JA, Ramsey MH. Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis-Mass Spectrometry. Anal Chem. 2016;88(4):2220–6.