HIGH SENSITIVITY LC-MS PROFILING OF ANTIBODY DRUG CONJUGATES WITH DIFLUOROACETIC ACID ION PAIRING AND A HIGH-COVERAGE PHENYL-BONDED STATIONARY PHASE

Significance of the topic

Protein subunit analysis by LC-MS is essential for detailed characterization of monoclonal antibodies and antibody drug conjugates. Traditional use of trifluoroacetic acid limits chromatographic throughput and suppresses MS signals. Difluoroacetic acid offers reduced ion pairing strength and lower ion suppression, resulting in improved chromatographic resolution and enhanced MS sensitivity for protein variants.

Objectives and study overview

This work aims to establish a robust high sensitivity LC-UV-MS platform for subunit profiling of antibodies and ADCs. Key goals include purification of difluoroacetic acid to remove metal adducts, adoption of a high coverage phenyl bonded stationary phase, and validation of method performance across various ADC formats and instrument configurations.

Methodology and instrumentation

• Ion pairing agent preparation purified reagent grade difluoroacetic acid by multiple distillations and quantified sodium and potassium by ICP
• Sample preparation included IdeS digestion and reduction of NIST mAb standard, a commercial SigmaMAb ADC mimic, and cysteine linked ADCs from Pfizer
• Chromatography employed Waters ACQUITY UPLC H Class or H Class Bio system with TUV or PDA detection
• Stationary phases comprised a 2.7 µm 2.1 x 50 mm BioResolve RP mAb Polyphenyl column and a 1.7 µm 2.1 x 150 mm ACQUITY BEH C4 300 Å column
• Mobile phases were 0.15 percent DFA in water (A) and 0.15 percent DFA in acetonitrile or 90/10 acetonitrile/IPA (B), with gradients from 15 to 55 percent B
• MS detection used a Xevo G2 XS QTof operated in sensitivity mode with MassLynx 4.1 and UNIFI 1.8 software; deconvolution was performed via MaxEnt1
• Robustness testing varied flow rate column temperature DFA concentration mass load by ±5 percent and included a 1000 injection lifetime study

Main results and discussion

• Use of 0.15 percent DFA yielded a fourfold increase in MS signal compared to 0.1 percent TFA
• Chromatographic resolution of subunits including oxidized and C terminal extended Fc 2 species was significantly improved
• Accurate drug to antibody ratio determination for auristatin conjugated antibody (DAR 4.2) and ADC mimic (DAR 3.5) matched HIC MS data
• Method robustness demonstrated relative standard deviations below 6 percent across stationary phase batches, DFA batches, LC systems, temperature, flow rate, and DFA concentration
• Adaptation for a highly hydrophobic ADC via increased column temperature highlighted method flexibility for challenging analytes

Benefits and practical applications

• High throughput subunit profiling with superior sensitivity and resolution
• Reliable detection of low level modifications and variants for detailed protein characterization
• Method compatibility with both UV and high resolution MS workflows supports QC and research environments
• Reproducible performance across different instruments and stationary phase batches

Future trends and possibilities

• Extension to analysis of bispecific antibodies, fusion proteins, and other biotherapeutics
• Integration of automated method development guided by real time MS feedback
• Evaluation of difluoroacetic acid in glycan profiling and intact protein analyses
• Development of new high coverage stationary phases for even more demanding separations

Conclusion

Purified difluoroacetic acid combined with a high coverage phenyl bonded column provides a robust LC-UV-MS platform for antibody and ADC subunit profiling. The method delivers significant gains in MS sensitivity, chromatographic resolution, and reproducibility suitable for advanced protein characterization in both research and industrial quality control settings.

Reference

1. Smith J Friese O Rouse J Nguyen J Lauber M Jayaraman P Characterization of Hydrophobic Monoclonal Antibodies and Antibody Drug Conjugates Presented at WCBP 2018 Washington DC United States January 28 31 2018
2. Nguyen JM Rzewuski S Walsh D Cook D Izzo G DeLoffi M Lauber MA Designing a New Particle Technology for Reversed Phase Separations of Proteins Waters Application Note PN 720006168EN 2018
3. Nguyen JM Kizekai L Walsh D Cook J Lauber MA A Novel Phenyl Bonded Phase for Improved Reversed Phase Separations of Proteins Waters Application Note PN 720006169EN 2018