Intact Analysis of Biopharmaceuticals by Hydrophobic Interaction/Reversed Phase 2D-LC/MS System

Importance of Topic

Hydrophobic Interaction Chromatography (HIC) is a critical technique for separating intact protein therapeutics based on surface hydrophobicity while maintaining their native conformation. It is routinely employed in downstream purification and increasingly in analytical workflows for monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs). However, the high salt concentrations required for HIC compromise compatibility with mass spectrometry (MS), limiting direct intact mass measurement of protein variants and drug-to-antibody ratio (DAR) profiling.

Study Objectives and Overview

This work presents a two-dimensional LC/MS (2D-LC/MS) strategy that integrates HIC in the first dimension with reversed-phase desalting and separation in the second. By applying multiple heart-cutting (MHC) between dimensions, the approach enables online desalting and subsequent time-of-flight (TOF) MS detection of intact biotherapeutics. Two proof-of-concept applications are described: forced oxidation of an mAb and analysis of a lysine-linked ADC.

Methodology and Instrumentation

Sample preparation involved dilution of stock salt solutions with water at pH 7 and 1:1 mixing with mobile phase B to control injection viscosity. Forced oxidation of the mAb was carried out with 1% tert-butyl hydroperoxide at 37 °C for 72 h. No deglycosylation was performed on the ADC sample.

The instrumentation configuration comprised:

• An Agilent 1290 Infinity 2D-LC system with 1260 Infinity II Bio-Inert LC for the HIC (1D) separation and 1290 Infinity II LC for the reversed-phase (2D) segment
• A 6224 TOF LC/MS with dual-nebulizer electrospray ionization for intact mass detection
• Agilent AdvanceBio HIC column (4.6 × 100 mm, 3.5 μm) at 25 °C and AdvanceBio RP-mAb C4 column (2.1 × 50 mm, 3.5 μm) at 80 °C
• Data analysis via Agilent OpenLAB CDS ChemStation and MassHunter BioConfirm software

Results and Discussion

Project 1 – Oxidized mAb

• The HIC chromatogram revealed a new oxidation peak at 12.05 min following t-BHP treatment.
• Heart-cutting into the RP dimension and TOF MS analysis indicated a +64 Da mass shift, consistent with four oxidation events on the intact antibody.

Project 2 – Lysine-Linked ADC

• HIC separation produced a broad, unresolved profile of positional isomers.
• Eight discrete retention windows were heart-cut, desalted on the RP column, and analyzed by TOF MS.
• Intact mass deconvolution identified ADC species with DAR values ranging from 0 to 8. Retention time correlated with increasing payload, and multiple heart-cuts revealed isomeric distributions and glycoform heterogeneity.

Benefits and Practical Applications

This 2D-LC/MS workflow provides a robust platform for intact mass characterization of mAbs and ADCs while preserving native structure. It enables accurate detection of minor variants, oxidation sites, and DAR profiling, supporting quality control, process development, and regulatory compliance in biopharmaceutical production.

Future Trends and Opportunities

Further advances may include integration with higher resolution mass analyzers, fully automated heart-cutting methods, novel HIC stationary phases for enhanced selectivity, and extension of the approach to emerging biotherapeutic formats such as bispecific antibodies and fusion proteins.

Conclusion

The combination of HIC with heart-cutting to reversed-phase LC and TOF MS overcomes the incompatibility of high-salt separations with MS detection. This 2D-LC/MS strategy delivers detailed intact mass profiling of protein therapeutics and ADCs, offering a powerful tool for routine characterization and release testing.

References

• Staples G. Analysis of monoclonal antibodies using MHC 2D-LC/MS. Agilent Technologies Application Note. 2014. (5991-6376EN)
• Agilent Technologies. AdvanceBio HIC and RP-mAb C4 columns, Technical Literature. 2019. (5994-1162EN)