Evaluation of a microfluidic electrophoresis device coupled to an Orbitrap mass spectrometer for the characterization of biotherapeutics proteins

Importance of the topic

The detailed analysis of biotherapeutic proteins is essential for ensuring their safety, efficacy, and consistency. Key characterization workflows—intact mass determination, sub-unit mass analysis, and peptide mapping—reveal molecular heterogeneity, post-translational modifications, and sequence integrity. Traditional LC-MS methods often require lengthy separations and significant sample preparation. Integrating microfluidic capillary electrophoresis (CE) with high-resolution Orbitrap mass spectrometry offers a fast, sensitive, and orthogonal approach that minimizes sample consumption and accelerates quality control in biopharmaceutical development.

Objectives and study overview

This work evaluated the performance of a chip-based electrophoresis device (ZipChip HR) coupled to Thermo Scientific Q Exactive Orbitrap instruments for three biotherapeutic characterization workflows: intact mass analysis, sub-unit mass analysis following IdeS proteolysis, and rapid peptide mapping. Specific goals included assessing speed, sensitivity, mass accuracy, and sequence coverage using the NIST monoclonal antibody (mAb) and the lysine-linked ADC trastuzumab emtansine.

Methodology

• Sample preparation for intact mass: simple dilution of NIST mAb and trastuzumab emtansine in water.
• Sub-unit analysis: IdeS digestion of intact mAb in Tris HCl, denaturation in 8 M guanidine, reduction with DTT, and buffer exchange using peptide kit.
• Peptide mapping: denaturation in guanidine HCl, reduction/alkylation with DTT/IAA, trypsin digestion at 37 °C for 30 min, quenching with formic acid, and dilution in microfluidic CE buffer.
• Separation conditions: ZipChip HR chip with field strengths of 500 V (intact), 220 V (sub-unit), and 400 V (peptide mapping).

Used instrumentation

• ZipChip HR microfluidic CE device (908 Devices) with 22 cm etched channel and integrated nano-ESI emitter.
• Thermo Scientific Q Exactive HF in high-mass-range (HMR) mode for intact mass analysis.
• Thermo Scientific Q Exactive Plus in protein mode for sub-unit mass analysis.
• BioPharma Finder 2.0 software for data processing, deconvolution, and peptide identification.

Main results and discussion

The microfluidic CE-Orbitrap platform delivered rapid separations and high-accuracy mass measurements across all workflows:

• Intact mass: Baseline separation of NIST mAb lysine variants (0K, +1K, +2K) in under one minute; identification of 15 glycoforms with mass errors below 14 ppm even at injection levels as low as 0.001 µg/µL.
• Sub-unit mass: Efficient separation of IdeS-derived subunits; detection of trastuzumab emtansine glycoforms G0F/G1F and G1F/G1F with average DAR values of 3.45 and 3.47 (mass error < 16 ppm), consistent with literature; lysine variant fragments identified with < 2.5 ppm error.
• Peptide mapping: Less than 10 min CE separation enabled MS/MS confirmation and achieved over 97% sequence coverage for both light and heavy chains of the NIST mAb.

Benefits and practical applications

• Significant reduction in analysis time compared to conventional LC-MS assays.
• Minimal sample consumption (sub-nanogram to low-nanogram range).
• High mass accuracy and sensitivity suitable for heterogeneity profiling of mAbs and ADCs.
• Streamlined workflows with minimal sample preparation for rapid QC and comparability studies.

Future trends and opportunities

Further refinement of microfluidic CE-MS platforms may include higher-throughput designs, automated sample handling, and integration with real-time data processing. Emerging applications could extend to vaccine antigen characterization, complex glycoproteins, and cell- and gene-therapy products. Advances in microchip materials and AI-driven data analysis promise to enhance resolution, sensitivity, and interpretation of complex biotherapeutic profiles.

Conclusion

The combination of ZipChip microfluidic electrophoresis with high-resolution Orbitrap mass spectrometry enables rapid, sensitive, and accurate characterization of intact proteins, sub-units, and peptides. This approach addresses the growing analytical demands of biotherapeutic development by delivering orthogonal separations, high sequence coverage, and low sample requirements in routine QC workflows.

References

• Marcoux J.F., et al. Protein Sci. 2015 Aug;24(8):1210–1223.