Characterization of Antibody‑Drug Conjugates Using 2D-LC and Native MS

Importance of the topic

Antibody–drug conjugates (ADCs) represent a rapidly growing class of targeted biotherapeutics that combine the selectivity of monoclonal antibodies with the potency of cytotoxic small molecules. A key quality attribute for ADC development is the drug‐to‐antibody ratio (DAR), which directly influences therapeutic efficacy and safety. Conventional 2D‐LC/MS methods using hydrophobic interaction chromatography (HIC) and reversed‐phase (RP) analysis often degrade ADCs under acidic and organic conditions, compromising accurate DAR determination and structural integrity.

Objectives and overview of the study

This application note describes a novel two‐dimensional liquid chromatography (2D‐LC) workflow coupled with native mass spectrometry (MS) for intact ADC characterization. The goals were to (1) develop an online HIC–SEC 2D‐LC setup that preserves native ADC structures, (2) accurately measure DAR values without sample degradation, and (3) streamline data acquisition and processing through a unified software platform.

Methodology and instrumentation

The workflow integrates three main steps:

• First‐dimension separation by HIC: AdvanceBio HIC column with a gentle ammonium sulfate gradient to resolve ADC species by hydrophobicity.
• Multiple heart‐cutting and desalting: Selective collection of HIC fractions via an Agilent multisampler and valve assembly, followed by online transfer.
• Second‐dimension separation and detection: Native conditions use AdvanceBio SEC for buffer exchange and desalting, coupled directly to an Agilent 6545XT AdvanceBio LC/Q‐TOF with extended mass range (up to m/z 30 000). Denaturing RP analysis (PLRP‐S) was also performed for comparison.

Instrumentation used

• Agilent 1290 Infinity II 2D‐LC system with multisampler, column thermostat, high‐speed pumps, and multidimensional valve drives
• Agilent AdvanceBio HIC (4.6 × 100 mm, 3.5 µm) and AdvanceBio SEC (4.6 × 300 mm, 1.9 µm) columns
• Agilent PLRP‐S reversed‐phase column for denaturing runs
• Agilent 6545XT AdvanceBio LC/Q‐TOF with Dual Jet Stream ESI source
• Agilent MassHunter Workstation 11.0 and BioConfirm 11.0 software for integrated 2D‐LC control, data acquisition, automated file splitting, and deconvolution

Main results and discussion

HIC separation of brentuximab vedotin revealed distinct peaks corresponding to DAR 0, 2, 4, 6, and 8 species, yielding an average DAR of ~3.7. Denaturing RP LC/MS analysis of isolated DAR-6 fractions showed multiple degradation products (half‐antibodies, light chains with payload truncation), confirming that acidic/organic solvents disrupt cysteine‐linked ADCs. In contrast, native SEC LC/MS preserved intact ADC complexes. Raw spectra (16+ to 27+ charge states) and deconvoluted masses matched expected values for DAR 0–6 with high mass accuracy (~10 ppm). DAR-8 species could not be reliably detected under native conditions due to low abundance and sensitivity limits.

Benefits and practical applications

• Preservation of intact ADC structures for accurate DAR measurement
• Reduction of sample degradation compared to denaturing methods
• Streamlined workflow with online buffering and desalting
• Unified software control minimizes manual intervention and data handling
• Applicability to quality control, stability studies, and biopharmaceutical development

Future trends and possibilities of use

Advancements may include higher‐sensitivity MS hardware for detecting very low‐abundance species (e.g., high‐DAR variants), integration of additional chromatographic modes (anion exchange, capillary SEC), and automation for high‐throughput biotherapeutic screening. Native 2D‐LC/MS can be extended to other protein–drug conjugates, antibody-oligonucleotide constructs, and noncovalent complexes to broaden its application in structural biology and bioanalysis.

Conclusion

A novel 2D‐LC/native MS workflow combining HIC, multiple heart‐cutting, and SEC under native conditions has been demonstrated for reliable ADC characterization. This approach overcomes degradation issues of traditional denaturing methods and delivers precise DAR values while preserving molecular integrity, making it a valuable tool for biopharmaceutical research and quality control.

References

1. Ross P.L.; et al. Physical and Chemical Stability of Antibody Drug Conjugates: Current Status. Journal of Pharmaceutical Sciences 2016, 105, 391–397.
2. Schneider S. Analysis of antibody drug conjugates using hydrophobic interaction chromatography with the Agilent 1290 Infinity II Bio LC System. Agilent Technologies application note 5994-2691EN, 2020.
3. van de Donk N.W.C.J.; Dhimolea E. Brentuximab Vedotin. mAbs 2012, 4(4), 458–465.
4. Wong D. Sensitive Native Mass Spectrometry of Macromolecules Using Standard Flow LC/MS. Agilent application note 5994-1739EN, 2020.
5. Wong D. Mass Spectrometric Characterization of Antibody-siRNA Conjugates using the Agilent 6545XT AdvanceBio LC/Q-TOF. Agilent application note 5994-2155EN, 2020.