Imaged capillary isoelectric focusing – Mass Spectrometry (iCIEF- MS) online coupling for polatuzumab vedotin charge heterogeneity analysis using native MS

Importance of the Topic

Antibody–drug conjugates (ADCs) such as polatuzumab vedotin represent a leading class of targeted biotherapeutics. Charge heterogeneity arising from drug conjugation and post-translational modifications can impact efficacy, safety and manufacturability. Sensitive characterization of these variants under near-native conditions is essential for quality control and biopharmaceutical development.

Objectives and Study Overview

This study aimed to develop and optimize an online coupling workflow between imaged capillary isoelectric focusing (iCIEF) and high-resolution Orbitrap mass spectrometry (MS) for charge variant analysis of cysteine-linked ADCs under native conditions. Polatuzumab vedotin served as the model ADC, and method performance was benchmarked using the NISTmAb standard.

Methodology and Instrumentation

The workflow integrated a CEInfinite iCIEF platform with a Thermo Scientific Orbitrap Exploris 240 mass spectrometer controlled via Chromeleon software.

• Sample preparation: Polatuzumab vedotin was buffer-exchanged and desalted to 1.5 mg/mL in aqueous solution with carrier ampholytes.
• iCIEF separation: Focusing steps applied a voltage ramp to 3000 V, followed by mobilization with 0.1% formic acid in water and a make-up liquid of 10 mM ammonium acetate/acetonitrile (9:1) at 3 μL/min.
• Mass spectrometry: Native intact MS acquisitions used optimized source settings, high resolution (30 000), and scan ranges up to m/z 8000 for accurate mass measurement.

Key Results and Discussion

The method successfully resolved and identified six main charge variant peaks of polatuzumab vedotin, including four acidic species, the principal peak, and two basic species.

• Native conditions without formamide preserved interchain interactions, albeit with reduced signal intensity that was compensated by the Orbitrap’s high sensitivity.
• Deconvoluted spectra revealed variant species with differing drug-to-antibody ratios (DAR), lysine truncations and N-glycoform distributions.
• Relative abundances of D2 and D4 glycoforms shifted across peaks, indicating conjugation and PTM contributions to charge heterogeneity.

Benefits and Practical Applications

The online iCIEF-MS platform under near-native conditions offers:

• High throughput and reproducible separation of ADC charge variants.
• Intact mass analysis preserving non-covalent interactions for accurate DAR and PTM profiling.
• Regulatory compliance via Chromeleon audit trails and integrated instrument control.

Future Trends and Potential Applications

Advances may include further sensitivity improvements, expanded native-MS coupling to other ADC formats, and automated data interpretation pipelines. Integration with orthogonal characterization techniques could deepen insight into structure–function relationships.

Conclusion

The developed iCIEF-MS workflow enables robust charge heterogeneity analysis of cysteine-linked ADCs under native conditions. It combines high-resolution separation, mass accuracy and software control to deliver detailed variant profiles essential for biopharmaceutical research and quality control.

Used Instrumentation

• CEInfinite iCIEF platform (Advanced Electrophoresis Solutions Ltd.)
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer with BioPharma option
• Thermo Scientific Chromeleon software for integrated control

References

1. Pharmaceuticals 2020, 13, 245; doi 10.3390/ph13090245
2. Analytica Chimica Acta 1297 (2024) 342349; doi 10.1016/j.aca.2024.342349
3. Analytical Biochemistry 680 (2023) 115312
4. Analytical Biochemistry 660 (2023) 114961