Hydrophobic Interaction Chromatography- Native Mode Separation of Bio-Therapeutic Workflows

Significance of the Topic

Hydrophobic interaction chromatography in native conditions offers a non-denaturing separation approach for complex proteins and bioconjugates such as monoclonal antibodies and antibody drug conjugates. By preserving biological activity and delivering high-resolution separation, this technique is critical for bio therapeutic characterization and quality control.

Objectives and Study Overview

The study evaluated a novel Agilent AdvanceBio HIC column for two applications: characterization of oxidized monoclonal antibody variants and determination of drug to antibody ratio of cysteine linked ADCs. It aimed to demonstrate improved speed, sensitivity and compatibility with high-salt mobile phases and mass spectrometry workflows.

Methodology

A fully porous 3.5 micron, 450Ã… pore AdvanceBio HIC stationary phase was employed under native conditions. Key liquid chromatographic parameters included:

• Column formats: 4.6x100 mm and 4.6x30 mm
• Mobile phases: 50 mM phosphate pH 7 (A), 2 M ammonium sulfate in phosphate pH 7 (B), isopropanol (C)
• Gradient elution from 45/50/5 to 75/0/25% (A/B/C)
• Flow rates 0.3–0.5 mL/min, injection volume 5 µL, 25 °C column temperature

Instrumentation

Analysis was performed on an Agilent 1260 Infinity II Bio-Inert HPLC system. Two-dimensional LC/MS experiments utilized an Agilent 1290 Infinity 2D-LC configured with a 6224 TOF mass spectrometer.

Main Results and Discussion

Oxidation of a NIST mAb with tert-butyl hydroperoxide produced multiple early-eluting variants. Two-dimensional LC/MS confirmed a +64 Da mass shift corresponding to oxidation of four methionine residues. For ADC analysis, the 4.6x30 mm column enabled an eight-minute separation versus 24 minutes on the standard column and accurately determined a DAR of 4.04.

Benefits and Practical Applications

• Native mode separation preserves biologically relevant structure and activity
• High throughput with faster run times
• Compatibility with high-salt mobile phases and mass spectrometry
• Effective characterization tool for mAbs and ADCs in research and QC

Future Trends and Opportunities

Advancements may include further integration of HIC with multi-dimensional LC/MS, novel stationary phase chemistries for enhanced selectivity, automated high-throughput workflows, and extension to other large biomolecules such as fusion proteins and viral vectors.

Conclusion

The AdvanceBio HIC column offers robust, high-resolution native separations for oxidized antibody variants and ADCs, yielding accurate mass characterization and DAR analysis with significantly reduced analysis times. Its compatibility with bio-inert systems and MS workflows makes it an attractive choice for therapeutic protein analytics.

References

1. Fekete S, Veuthey JL, Beck A, Guillarme D. J Pharm Biomed Anal. 2016, 130, 3–18.
2. Staples G. Agilent Technologies Application Note 5991-6376EN, 2014.
3. Coffey A, Kondaveeti S. Agilent Technologies Application Note 5994-0149EN, 2018.