Analysis of Monoclonal Antibodies and Antibody-Drug Conjugates Using New Hydrophobic Interaction Chromatography (HIC) Columns

Significance of the topic

Monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) are cornerstone biotherapeutics in oncology and beyond. Their complex structures and susceptibility to aggregation, fragmentation, oxidation and drug conjugation demand robust analytical techniques. Hydrophobic interaction chromatography (HIC) has emerged as a key orthogonal method for high‐resolution separation of mAb variants, ensuring drug safety, efficacy and batch‐to‐batch consistency.

Objectives and overview

This study introduces a new series of Thermo Scientific MAbPac HIC columns featuring polyamide, amide and butyl ligand chemistries. The aim was to demonstrate their performance in resolving mAb aggregates, Fc/Fab fragments, oxidized species and ADC mimics under a variety of mobile-phase conditions.

Methodology and used instrumentation

UltiMate 3000 BioRS system equipped with SR-3000 solvent rack, LPG-3400RS pump, WPS-3000TBRS autosampler, TCC-3000RS column compartment and VWD-3400RS detector was used. MAbPac HIC-10 (4.6×100 mm), HIC-20 (4.6×100 mm and 4.6×250 mm) and HIC-Butyl (4.6×100 mm) columns were evaluated. Mobile phases combined 1.5–2 M ammonium sulfate or 100 mM sodium phosphate, pH 7.0, with isopropanol gradients (5–20 %) to modulate hydrophobic interactions.

Main results and discussion

• Aggregate separation: All three columns resolved monomer from high‐order aggregates with sharp peaks and recoveries >95 % using optimized salt and organic modifiers.
• Fragment profiling: Papain-digested mAb fragments (Fab, Fc) were baseline separated; polyamide and amide chemistries provided distinct hydrophilic/hydrophobic discrimination of oxidized and charge variants.
• Oxidation analysis: HIC-20 columns differentiated untreated mAb from peroxide or AAPH-oxidized forms, with variant peaks clearly resolved under 5 % IPA in phase A and 20 % IPA in phase B at 25 °C.
• ADC mimic characterization: Cysteine-linked ADC analogs (DAR 0-8) eluted in order of increasing hydrophobicity on the Butyl column. Higher‐loaded species showed longer retention, enabling quantification of drug load distribution.

Benefits and practical applications

The new MAbPac HIC columns offer:

• High resolution of critical quality attributes (aggregates, fragments, oxidized forms, DAR species).
• Fast method development via simple gradient adjustments and organic modulator addition.
• Compatibility with standard biocompatible HPLC systems and direct integration into QA/QC workflows.

Future trends and opportunities

Continued advances may include smaller particle HIC phases for ultrahigh throughput, coupling with mass spectrometry for on‐line identification of variants, and tailored ligand chemistries for novel bioconjugates. Machine-learning-driven method optimization and microflow HIC formats are poised to accelerate characterization of next‐generation biotherapeutics.

Conclusion

Thermo Scientific MAbPac HIC columns with polyamide, amide and butyl ligands deliver robust, high‐efficiency separations of mAb heterogeneities and ADC variants. Their versatile selectivity and ease of gradient tuning support critical analytical tasks in biopharmaceutical development and quality control.

Reference

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