Analyses of Antibody Drugs Using Ultra HighPerformance Liquid Chromatography

Significance of the Topic

Biopharmaceuticals such as monoclonal antibodies and antibody–drug conjugates play a key role in modern therapeutics but require stringent control of structural variants and impurities to ensure safety and efficacy. Liquid chromatography is a cornerstone method for separating and quantifying aggregates, charge variants, and conjugation heterogeneity. The Nexera XS inert UHPLC system enhances robustness in high salt environments and reduces undesirable sample–stationary phase interactions, improving reproducibility and accuracy in quality control.

Objectives and Study Overview

This study demonstrates analytical workflows for evaluating mAb and ADC quality attributes using an inert UHPLC platform. Key aims include separation of aggregates, charge isoforms, and drug–to–antibody ratio variants under optimized chromatographic conditions. Comparative assessments highlight method adjustments to overcome hydrophobic interactions and maximize resolution.

Methods and Instrumentation Used

Liquid chromatography modalities included:

• Size Exclusion Chromatography (SEC) for aggregate and degradate analysis
• Ion Exchange Chromatography (IEX) for acidic and basic charge variant profiling
• Hydrophobic Interaction Chromatography (HIC) for drug–to–antibody ratio determination

Chromatographic conditions were tailored via column selection and mobile phase composition. High corrosive salt concentrations were tolerated by the inert flow path. Gradient and isocratic modes were applied as needed to optimize peak resolution.

Instrumentation Used

• Nexera XS inert ultra high performance liquid chromatograph with inert flow path
• TSKgel series columns (UP-SW3000 for SEC, CM-STAT for IEX, Butyl-NPR for HIC)
• SPD-M40 UV detector (280 nm)
• TORAST-H inert glass vials

Main Results and Discussion

• SEC achieved baseline separation of mAb multimer, monomer, and degradates within 5 minutes under isocratic phosphate buffer containing sulfate
• In ADC analysis, addition of 15% 2-propanol suppressed hydrophobic interaction and sharpened peaks, enabling clear aggregate profiles
• IEX separated multiple charge variants in three mAb samples using a MES buffer gradient to resolve acidic and basic isoforms
• HIC resolved non-conjugated and ADC species by drug load, distinguishing DAR 0 through 6 in a single run

Benefits and Practical Applications

• Rapid and reproducible assays support high-throughput quality control workflows
• Stable performance under high salt mobile phases reduces maintenance and downtime
• Versatile method adaptation allows tailored analysis of diverse biotherapeutics

Future Trends and Potential Uses

The adoption of inert UHPLC systems is expected to extend to other complex biologics, including fusion proteins and glyco-engineered compounds. Integration with mass spectrometry and automated method development software will streamline impurity profiling. Emerging materials with enhanced selectivity and robustness will further improve precision in biomolecule characterization.

Conclusion

The optimized chromatographic strategies presented here offer efficient, reproducible, and high-resolution analyses of critical quality attributes in antibody drugs and ADCs. The Nexera XS inert platform demonstrates the ability to handle challenging mobile phases and minimize sample interactions, supporting rigorous biopharmaceutical quality control.

References

1. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products International Council for Harmonisation 2001
2. Ministry of Health Labour and Welfare PFSD/ELD Notification No 571 2001
3. Biopharmaceutical Handbook 4th Edition Jiho Inc
4. T Arakawa et al Solvent Modulation of Chromatography Journal of Japanese Biochemical Society 80 1 2021