Analysis of Monoclonal Antibodies

Significance of the Topic

Monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) have become essential biotherapeutics in oncology and immunology. Precise characterization of mAb variants, post-translational modifications and drug-to-antibody ratio (DAR) in ADCs is critical for safety and efficacy. Hydrophobic interaction chromatography (HIC) offers selective separation of protein isoforms and ADC species, but its compatibility with mass spectrometry (MS) is limited by nonvolatile salts.

Objectives and Article Overview

This work describes a two-dimensional liquid chromatography approach that combines HIC in the first dimension with reversed-phase (RP) chromatography in the second dimension, coupled on-line to time-of-flight MS. The goal is to enable multiple heart-cutting (MHC) from HIC separations, effective desalting and high-resolution MS detection of mAbs and ADCs.

Methodology and Instrumentation

Instrumental platform:

• Agilent 1260 Infinity Bio-inert quaternary pump for HIC (1D)
• Agilent 1290 Infinity binary pump for RP (2D)
• Agilent 1260 Bio-inert autosampler with thermostat
• Agilent 1290 thermostatted column compartment and valve drive with multiple heart-cutting upgrade
• Agilent 6224 TOF LC/MS with dual-nebulizer ESI source

Chromatographic conditions:

• HIC stationary phase: silica- or polymer-based columns, 4.6×100 mm; mobile phases containing phosphate buffer and nonvolatile salt (ammonium tartrate optimized for MS compatibility)
• RP stationary phase: AdvanceBio RP-mAb C4, 2.1×50 mm; mobile phases with formic/acetic acid modifiers and organic solvents
• Heart-cutting via 2-position/4-port duo valve with six 40 µL loops to transfer selected peaks from 1D to 2D
• Flow rates: 0.4 mL/min (1D), 0.2 mL/min (2D); column temperatures: 25 °C (HIC), 80 °C (RP)

Main Results and Discussion

1. mAb A analysis:
Multiple low-abundance satellite peaks were resolved by HIC and heart-cut to RP-MS. Deconvoluted masses revealed truncated variants (up to –9.9 kDa) and a hydrophobic isomer with identical mass.

2. Oxidized mAb B:
Forced oxidation generated a peak shifted by –0.5 min in HIC. MS of heart-cuts showed a +64 Da mass increase, consistent with four methionine oxidations.

3. Lys-linked ADC (mAb C):
HIC alone produced a broad unresolved profile due to multiple positional isomers. Eight heart-cuts transferred to RP-MS allowed deconvolution of DAR species from 0 to 8 and identification of linker-only species.

These results demonstrate that HIC/RP 2D-LC/MS yields high desalting efficiency, sensitive MS signal at low sample load (20 µg), and flexibility to accommodate extended 2D gradients.

Benefits and Practical Applications

• Unambiguous mass measurement of mAb variants, oxidation products and ADC species
• Online desalting and separation without offline sample preparation
• Ability to investigate minor components (<1 % abundance) and unknown peaks
• Reduced sample consumption and improved throughput in biopharmaceutical QC and R&D

Future Trends and Opportunities

• Integration of top-down proteomics workflows by coupling HIC/RP 2D-LC with high-resolution MS/MS
• Automation of heart-cut scheduling and real-time data-driven peak selection
• Expansion to other non-MS compatible separations (e.g., ion-exchange) in multidimensional platforms
• Adoption for characterization of emerging modalities such as bispecific antibodies and fragment-drug conjugates

Conclusion

The presented HIC/RP 2D-LC/MS strategy overcomes the salt incompatibility of HIC separations for mass spectrometry. The multiple heart-cutting approach enables detailed characterization of mAb heterogeneity, oxidative modifications and ADC DAR profiles, offering a robust tool for biotherapeutic analysis.

References

1. Buckenmaier S. Agilent 1290 Infinity 2D-LC Solution for Multiple Heart-Cutting. Agilent Technologies Technical Overview, publication number 5991-5615EN, 2015.
2. Xiu L. et al. Effective Protein Separation by Coupling HIC and RP for Top-down Proteomics. Anal. Chem. 2014, 86, 7899–7906.