High Salt—High Reproducibility - Analysis of antibody drug conjugates using hydrophobic interaction chromatography with the Agilent 1290 Infinity II Bio LC System

Importance of the Topic

The drug-to-antibody ratio (DAR) of antibody-drug conjugates (ADCs) is a critical quality attribute that directly impacts therapeutic safety and efficacy. Hydrophobic interaction chromatography (HIC) under native conditions is the reference method to resolve ADC species with different drug loads. However, HIC mobile phases often contain high concentrations of corrosive salts that can damage standard LC systems and cause drifting baselines when using less pure reagents.

Objectives and Study Overview

This study demonstrates the use of the Agilent 1290 Infinity II Bio LC System to determine the DAR of brentuximab vedotin by HIC. Key goals include: accurate DAR calculation, evaluation of retention time and area reproducibility, and demonstration of software-based baseline correction to accommodate impure ammonium sulfate.

Methodology and Instrumentation

The Agilent 1290 Infinity II Bio LC System features an iron-free flow path (MP35N alloy) to resist corrosion by high-salt buffers. System configuration included:

• High-Speed Pump and dedicated solvent tables for concentrated salts
• Multisampler with cooling and seal wash
• Multicolumn Thermostat for stable temperature control
• Variable Wavelength Detector (280 nm, 2 µL flow cell)
• Agilent OpenLab CDS software with “Blank Subtraction” feature
• AdvanceBio HIC column (4.6 × 100 mm, 3.5 µm)

Mobile phase A was 1.5 M ammonium sulfate in 50 mM phosphate buffer (pH 7); mobile phase B was 50 mM phosphate buffer (pH 7) with 20 % isopropanol. A linear gradient from 30 % to 100 % B over 30 min at 0.4 mL/min and 25 °C was used. Samples of brentuximab vedotin (100 mg/mL total formulation) were injected (15 µL) with needle wash to prevent salt buildup.

Main Results and Discussion

HIC resolved five major peaks corresponding to antibody species carrying 0, 2, 4, 6, and 8 drug molecules (D0–D8). Integration yielded a DAR of 3.7, consistent with literature values. Seven replicate injections showed excellent reproducibility: retention time RSD < 0.081 % and area RSD < 0.282 %. The “Blank Subtraction” feature effectively removed baseline drift caused by less pure ammonium sulfate, ensuring accurate peak integration.

Benefits and Practical Applications

• Robust DAR determination under native, nondenaturing conditions for ADC quality control
• Corrosion-resistant hardware minimizes maintenance and prolongs instrument lifespan
• High reproducibility reinforces confidence in critical bioanalytical data
• Software-based baseline correction allows use of economical reagents without compromising results

Future Trends and Potential Applications

Advances may include further automation of HIC workflows, real-time DAR monitoring, application to diverse bioconjugates, and enhanced software algorithms for baseline correction. Integration with mass spectrometry and multidimensional separations may expand analytical capabilities for complex biopharmaceuticals.

Conclusion

The Agilent 1290 Infinity II Bio LC System, combined with Agilent OpenLab CDS, provides a highly reproducible and corrosion-resistant platform for HIC-based DAR analysis of cysteine-linked ADCs. The system’s hardware and software features ensure reliable data generation and streamlined workflows for biopharmaceutical research and quality control.

References

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