Novel Hydrophobic Interaction HPLC Columns

Importance of Hydrophobic Interaction Chromatography for Monoclonal Antibodies

Hydrophobic interaction chromatography (HIC) plays a critical role in the detailed characterization of monoclonal antibodies (mAbs) and related biologics. These large, heterogeneous molecules undergo various post-translational modifications and conjugations that influence their stability, efficacy and safety. As a native-compatible, orthogonal technique to cation exchange and size-exclusion chromatography, HIC enables resolution of subtle hydrophobicity differences tied to glycosylation, oxidation, deamidation, aggregation and drug-to-antibody ratio in antibody drug conjugates (ADCs).

Objectives and Overview

This study examines the Thermo Scientific MAbPac HIC column series—HIC-10, HIC-20 and HIC-Butyl—for broad selectivity in mAb analysis. The goal is to demonstrate column performance for intact antibodies, fragments, aggregates, oxidative variants and ADCs with varying drug loads, highlighting resolution, bio-compatibility and method robustness.

Methodology and Instrumentation

MAbPac HIC separations employ a high-salt loading buffer to promote reversible binding of proteins by hydrophobic interactions, followed by a decreasing salt gradient to elute species in order of hydrophobicity. Key parameters include:

• Stationary phases: proprietary polyamide or amide chemistries on spherical silica (5 µm, 1,000 Å) and hydrophilic butyl polymer
• Mobile phase A: ammonium sulfate salts (1.5–2 M) with phosphate at pH 7.0, optionally spiked with isopropanol
• Mobile phase B: low-salt phosphate buffers, with variable organic modifiers
• Gradient durations from 7 to 35 min and flow rates of 0.5–1.5 mL/min
• Detection by UV absorbance at 280 nm

Použitá instrumentace

• Thermo Scientific MAbPac HIC-10, HIC-20 and HIC-Butyl columns (5 µm; formats 4.6×100 mm and 4.6×250 mm)
• HPLC system capable of up to 8,000 psi pressure
• Syringe or autosampler injection of 5–20 µL sample volumes
• Guard columns and holders for column protection

Main Results and Discussion

Chromatographic performance was demonstrated across multiple applications:

• ADC separations: Cysteine-conjugated ADC mimics with DARs 0–8 were baseline-resolved on HIC-Butyl, confirming linear retention shifts with increasing drug load.
• Protein mixtures: Myoglobin, ribonuclease A, lysozyme and α-chymotrypsinogen A were separated with sharp, symmetric peaks on HIC-10, illustrating broad protein compatibility.
• Aggregate analysis: HIC-10 distinguished mAb monomer, hydrophilic variants and higher-order aggregates in a single run, offering an alternative to SEC for aggregation profiling.
• Fragment resolution: Papain-digested mAb fragments (Fab and Fc) and their variants were clearly resolved on HIC-20, enabling domain-specific variant mapping.
• Oxidized variants: HIC-20 separated unmodified mAb from H2O2 and AAPH-oxidized species without additional sample preparation.

Benefits and Practical Applications

The MAbPac HIC family delivers:

• High selectivity across diverse mAb formats (intact, fragments, ADCs, bispecifics)
• Native-compatible conditions preserving protein conformation for downstream assays
• Robust, high-efficiency columns with wide pH and solvent compatibility
• Orthogonal data supporting CEX, SEC and reverse-phase analyses in QC and method development

Future Trends and Opportunities

Emerging directions include coupling HIC to mass spectrometry for direct variant identification, development of next-generation stationary phases with tunable hydrophobicity, integration into automated high-throughput platforms, and application of machine learning for method optimization. Single-use HIC cartridges and inline online monitoring may further streamline bioprocess analytics.

Conclusion

Thermo Scientific MAbPac HIC columns provide a versatile, high-resolution platform for comprehensive hydrophobicity-based characterization of mAbs and related biologics. Their complementary selectivity, bio-compatibility and robustness make them an essential tool in biopharmaceutical R&D and quality control.