Intact Analysis Using Hydrophobic Interaction - Agilent BioHPLC Columns Application Compendium

Significance of the topic

Hydrophobic interaction chromatography (HIC) plays a critical role in the characterization of intact proteins, especially therapeutic monoclonal antibodies (mAbs). By preserving native protein conformation and exploiting differences in surface hydrophobicity, HIC enables the detection and quantification of post-translational modifications (PTMs), aggregates, and conjugated species without denaturing the analyte. This approach supports quality control in biopharmaceutical development, ensuring product efficacy and safety.

Objectives and overview of the article

The article introduces the Agilent AdvanceBio HIC column and its application with the 1260 Infinity II Bio-inert liquid chromatography system to separate oxidized mAb variants from native forms. Key goals include demonstrating improved selectivity for methionine oxidation products, outlining optimized chromatographic conditions, and illustrating the method’s reproducibility for routine analytical workflows.

Methodology and instrumentation

• Chromatographic principle: Proteins bind to a weakly hydrophobic stationary phase in a high-salt mobile phase and elute under a decreasing salt gradient ("salting out" effect).
• Stationary phase: AdvanceBio HIC column, fully porous silica, 3.5 µm, 450 Å pore size; dimensions 4.6 × 100 mm for high resolution or 4.6 × 30 mm for rapid separations.
• Mobile phases: A – 50 mM sodium phosphate, pH 7.0; B – 2 M ammonium sulfate in phosphate buffer; C – isopropanol (optional for elution of highly hydrophobic species).
• Gradient: Typical gradient from 50 % A/50 % B to 100 % A over 20 minutes, flow rate 0.3–0.5 mL/min, column temperature 25 °C.
• Instrumentation: Agilent 1260 Infinity II Bio-inert LC system with quaternary pump, refrigerated multisampler, bio-inert column compartment, and diode array detector.
• Sample oxidation: NISTmAb treated with 0.2 % tert-butyl hydroperoxide (t-BHP) or hydrogen peroxide (H₂O₂) to generate surface-accessible and buried methionine oxidation products, followed by buffer exchange.

Main results and discussion

• Separation of native and oxidized species: Untreated NISTmAb elutes as a single peak, whereas oxidized samples display earlier-eluting shoulders and distinct peaks corresponding to mono- and di-oxidized variants.
• Effect of oxidant: t-BHP generates multiple oxidation products with moderate shifts in retention, while H₂O₂ yields more complete oxidation and fewer distinct species.
• Optimization: A shallower salt gradient and reduced flow rate improved resolution, enabling baseline separation of up to six oxidized forms within 40 minutes.
• Time-course monitoring: Progressive broadening and retention shifts observed over reaction time indicate stepwise oxidation of surface-accessible followed by buried methionines.

Benefits and practical applications

HIC methods maintain protein bioactivity, facilitating downstream potency assays on collected fractions. They complement size exclusion, ion exchange, and reversed-phase techniques by offering unique selectivity based on surface hydrophobicity. Key applications include aggregate removal, antibody-drug conjugate drug-to-antibody ratio determination, bispecific antibody purity assessment, and stability studies under stress conditions.

Future trends and potential uses

• Integration with mass spectrometry for intact mass mapping and confirmation of modification sites.
• Automation and high-throughput screening in process development and QA/QC laboratories.
• Expansion to other biotherapeutic modalities, such as fusion proteins and enzymes, to characterize diverse PTMs.
• Data-driven method modeling and machine learning to predict retention behavior from sequence and structural features.

Conclusion

The Agilent AdvanceBio HIC column paired with a bio-inert LC system provides robust, reproducible separation of intact mAb oxidation variants under native conditions. Optimized salt gradients and column chemistries enable high resolution of closely related species, supporting critical quality attribute analysis in biopharmaceutical development.

References

1. Zhang, Y.; Martinez, L.; Woodruff, B.; Goetze, A.; Bailey, R.; Pettit, D.; Balland, A. Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency. Anal. Chem. 2008, 80(18), 7022–7028.
2. Gaza-Bulseco, G.; et al. Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2008, 850(1–2), 285–294.
3. Boyd, D.; Kaschak, T.; Yan, B. HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2011, 879(13–14), 955–960.
4. Fekete, S.; Guillarme, D.; Beck, A.; Veuthey, J. Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products. J. Pharm. Biomed. Anal. 2016, 130, 3–18.
5. Liu, D.; Gokarn, Y.; Harrison, R.K. Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation. Biochemistry 2008, 47(18), 5088–5100.