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

Significance of the topic

The rapid growth of biotherapeutics demands analytical methods that deliver high sensitivity, low sample consumption and fast turnaround. Combining microfluidic capillary electrophoresis with high-resolution mass spectrometry offers an orthogonal, efficient approach to intact mass, sub-unit and peptide mapping analyses critical for ensuring safety, efficacy and product consistency.

Objectives and study overview

This study evaluates the performance of a chip-based electrophoresis device (ZipChip HR) coupled to Orbitrap-based mass spectrometers (Q Exactive HF and Plus) across three workflows:

• Intact mass analysis of monoclonal antibodies and antibody–drug conjugates
• Sub-unit analysis following IdeS digestion
• Peptide mapping by rapid tryptic digest

Methodology and instrumentation

Samples: NIST monoclonal antibody (mAb) and trastuzumab emtansine.
Sample preparation:

• Intact: dilution in water
• Sub-unit: IdeS digestion, denaturation, reduction, buffer exchange
• Peptide mapping: denaturation, reduction/alkylation, trypsinization (30 min), quench and dilution

Microfluidic electrophoresis:

• ZipChip HR (908 Devices) with field strengths 500 V (intact), 220 V (sub-unit) and 400 V (peptides)

Mass spectrometry:

• Thermo Scientific Q Exactive HF in high mass range for intact analysis
• Q Exactive Plus in protein mode for sub-unit
• Q Exactive HF in standard mode for peptide mapping

Data analysis: Thermo Scientific BioPharma Finder 2.0 software.

Results and discussion

Intact mass analysis:

• Baseline separation of NIST mAb lysine variants at 0.65 nL injection; mass error <10 ppm
• Identification of trastuzumab emtansine glycoforms G0F/G1F and G1F/G1F with average DAR 3.46 and 3.47; mass error <16 ppm

Sub-unit analysis:

• IdeS-derived light and heavy chains separated within minutes
• High mass accuracy (<2.5 ppm) allowed unambiguous variant assignment

Peptide mapping:

• Sequence coverage >97% for both chains after a 10 min separation
• MS/MS confirmation of peptide identities

Practical benefits and applications

This approach delivers:

• Fast separation (<40 s for ADC forms)
• Low sample consumption (sub-nanogram injections)
• High sensitivity and accuracy for QC and R&D workflows in biopharmaceutical development

Future trends and opportunities

Advancements may include:

• Integration of automated sample prep with microfluidic separation
• High-throughput proteoform profiling for process monitoring
• Coupling with ion mobility and data-independent acquisition for deeper characterization

Conclusion

The ZipChip-Orbitrap platform provides a robust, rapid and sensitive solution for intact mass, sub-unit and peptide mapping workflows. Its low sample requirements and high resolving power make it well suited for modern biotherapeutic analysis and quality control.

Reference

1. Marcoux et al., Protein Science, 2015 Aug;24(8):1210-1223