MAbPac HIC-Butyl Column High resolution HIC column for monoclonal antibody analysis

Significance of the Topic

Hydrophobic interaction chromatography (HIC) plays a critical role in the characterization of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs), offering an orthogonal approach to ion exchange and size-exclusion techniques while preserving protein structure and biological activity. By separating proteoforms based on hydrophobicity under non-denaturing conditions, HIC supports detailed analysis of biochemical modifications, purity, and heterogeneity, which are essential for ensuring therapeutic safety and efficacy.

Objectives and Study Overview

This work evaluates the performance of a polymer-based hydrophilic interaction chromatography column, the Thermo Scientific™ MAbPac™ HIC-Butyl, for high-resolution separation of mAbs, ADC mimics, and mAb fragments. Key aims include comparing selectivity with existing HIC columns, demonstrating capability for resolving cysteine- and lysine-linked ADC variants, and assessing fragment analysis following enzymatic digestion.

Methodology

The separation principle relies on controlled binding of proteins to a weakly hydrophobic butyl-functionalized stationary phase in high-salt buffer, followed by elution through salt reduction or solvent variation. Various sample types were tested:

• Mixed standard proteins and an intact mAb
• Cysteine-conjugated ADC mimic (DAR 0–8)
• Lysine-conjugated ADC mimic
• Papain-generated Fab and Fc fragments

Chromatographic conditions (gradient, flow rate, temperature) were optimized for each application to maximize resolution.

Used Instrumentation

• Column: MAbPac HIC-Butyl, 5 µm hydrophilic polymer, butyl ligand, non-porous (4.6 × 100 mm analytical format)
• Mobile phases: Ammonium sulfate/phosphate buffers (1.5–2 M salt) with optional isopropanol content
• Flow rates: 0.5–1.0 mL/min (up to 2.0 mL/min for guard format)
• Column temperature: 25–35 °C
• Detection: UV absorbance at 280 nm

Main Results and Discussion

High-resolution separation of small proteins (e.g., myoglobin, ribonuclease A, lysozyme) and a model mAb achieved sharp peaks and clear baseline separation within 20 minutes. Cysteine-linked ADC mimics (DAR 0–8) displayed well-resolved species, highlighting the column’s sensitivity to incremental hydrophobicity changes. Lysine-conjugated ADCs, inherently more heterogeneous, also showed distinct clusters of drug-loaded variants. Papain digestion yielded clear separation of Fab and Fc fragments as well as their microheterogeneities, demonstrating utility for fragment-level analysis. Column-to-column reproducibility was confirmed through stringent manufacturing and qualification protocols.

Benefits and Practical Applications of the Method

• Orthogonal characterization tool complementary to cation exchange and size exclusion chromatography
• Non-denaturing conditions preserve mAb functionality for downstream bioassays
• High selectivity for ADC variants enabling drug-to-antibody ratio assessment
• Broad pH stability (2–12) and compatibility with up to 50% organic solvents
• Low sample carryover and robust column packing for reliable routine operation

Future Trends and Potential Uses

Advances in HIC stationary phases may further improve resolution of complex biotherapeutics, including multispecific antibodies and novel conjugates. Integration with high-resolution mass spectrometry promises direct structural elucidation of separated species. Continued development of biocompatible polymers and tailored ligand chemistries will expand applications to viral vectors, protein–protein complexes, and real-time process monitoring in biomanufacturing.

Conclusion

The MAbPac HIC-Butyl column delivers high efficiency and reproducible hydrophobic interaction separations for mAbs, ADC mimics, and antibody fragments under non-denaturing conditions. Its complementary selectivity, broad operational range, and low carryover support comprehensive characterization workflows critical to therapeutic development and quality control.