Enhancing Subunit-Level Profiling of mAbs and ADCs with MS- Quality Difluoroacetic Acid

Significance of the Topic

Monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) are increasingly critical in biotherapeutics, demanding precise characterization at the subunit level. Reliable liquid chromatography–mass spectrometry (LC-MS) profiling of reduced and digested antibody fragments reveals structural variants, post-translational modifications, and drug–antibody ratios (DAR). Improving resolution, throughput, and sensitivity enhances quality control, accelerates development, and supports regulatory compliance in biopharmaceutical workflows.

Objectives and Study Overview

This study aimed to develop a high-performance LC-MS platform that overcomes limitations of traditional trifluoroacetic acid (TFA) mobile phases and conventional reversed-phase columns. Specific goals included:

1. Purification of difluoroacetic acid (DFA) to LC-MS grade to minimize metal contaminants.
2. Integration of DFA with a novel superficially porous phenyl column to boost chromatographic resolution and protein recovery.
3. Optimization of MS parameters for low adduct formation and enhanced sensitivity.
4. Validation of method robustness and reproducibility for mAb subunits and hydrophobic ADC fragments.

Methodology and Used Instrumentation

Samples of reduced, IdeS-digested NIST mAb subunit standard and Pfizer-manufactured ADCs were analyzed. DFA reagent was purified through multiple distillation steps, yielding low metal levels confirmed by ICP-MS (sodium and potassium <20 ppb). Chromatographic separations employed:

• Waters BioResolve RP mAb Polyphenyl column (2.7 μm, 2.1×150 mm, 450 Å) or ACQUITY BEH C4 (1.7 μm, 300 Å) for comparison.
• Mobile phases using 0.15% DFA in water (A) and in acetonitrile or isopropanol blends (B), at flow rates up to 0.6 mL/min and temperatures of 70–80 °C.

Instrumentation included an ACQUITY UPLC H-Class Bio system with TUV detector, coupled to a Xevo G2-XS QToF mass spectrometer. MS settings were fine-tuned to reduce metal adducts and acquire deconvoluted spectra with MassLynx and UNIFI software.

Main Results and Discussion

Key findings:

• DFA versus TFA: 0.1% DFA delivered approximately fourfold higher MS signal intensity for protein subunits due to weaker ion pairing and reduced suppression.
• Chromatographic performance: The phenyl column with DFA achieved sharper peaks, higher resolution of oxidized and aglycosylated light chain fragments, and accelerated run times compared to TFA methods.
• Metal contamination: ICP-MS confirmed that distilled DFA matched commercial LC-MS quality of formic and trifluoroacetic acids, eliminating interference in mass spectra.
• Method robustness: Qualification studies varying temperature, flow rate, organic content, DFA concentration, column batches, and systems produced relative standard deviations below 6% for retention times and peak areas.
• ADC profiling: Subunit separations of a cysteine-linked ADC demonstrated reliable DAR determination (average DAR = 4.2) with excellent repeatability across 1000 injections.

Benefits and Practical Application of the Method

By combining highly purified DFA and the polyphenyl stationary phase, the new platform affords:

• Enhanced MS sensitivity for low-abundance modifications and payload-linked fragments.
• Improved chromatographic resolution and throughput for mAb/ADC subunit analyses.
• Greater protein recovery and reduced sample degradation at lower temperatures.
• Robust performance suitable for routine QA/QC, process development, and regulatory submissions.

Future Trends and Potential Applications

The success of DFA-based mobile phases and phenyl-bonded superficially porous particles suggests potential expansion to other biotherapeutics, including peptide conjugates and next-generation biologics. Further integration with ultrahigh-throughput systems and automated workflows may support large-scale screening, stability studies, and in-depth structural characterization using data-independent acquisition and high-resolution MS.

Conclusion

The developed LC-MS platform leverages LC-MS-grade DFA and a novel polyphenyl column to achieve unprecedented resolution, sensitivity, and robustness in subunit profiling of mAbs and ADCs. This approach addresses longstanding challenges with TFA-based methods, enabling detailed characterization of therapeutic antibodies and advancing analytical capabilities in biopharmaceutical research and quality control.

References

• Smith J., Friese O., Rouse J., Nguyen J., Lauber M. & Jayaraman P. Characterization of Hydrophobic Monoclonal Antibodies and Antibody-Drug Conjugates. WCBP 2018, Washington D.C.
• Nguyen J.M., Rzewuski S., Walsh D., Cook D., Izzo G., DeLoffi M. & Lauber M.A. Designing a New Particle Technology for Reversed-Phase Separations of Proteins. Waters Application Note, 2018.
• Fekete S., Veuthey J. & Guillarme D. New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications. J. Pharm. Biomed. Anal. 69, 9–27 (2012).
• Nguyen J.M., Kizekai L., Walsh D., Cook J. & Lauber M.A. A Novel Phenyl Bonded Phase for Improved Reversed-Phase Separations of Proteins. Waters Application Note, 2018.