Agilent AdvanceBio Desalting-RP Cartridges for Online Desalting in 2D‑LC/MS mAb Analysis

Importance of the Topic

Monoclonal antibodies (mAbs) are complex biotherapeutics that undergo diverse chemical and enzymatic modifications, resulting in heterogeneity such as charge variants, glycoforms, and sequence variants. Accurate physicochemical characterization is essential to ensure product safety, quality, and efficacy. Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) with online desalting addresses challenges posed by nonvolatile salts in first-dimension separations and enables seamless MS detection of mAb variants.

Objectives and Study Overview

This application note demonstrates a 2D-LC/MS workflow using Agilent AdvanceBio Desalting-RP cartridges in the second dimension. The first dimension employs either weak cation exchange (IEX) for charge variant profiling or Protein A affinity capture for mAb enrichment. The study evaluates performance on innovator and biosimilar mAbs, assessing separation efficiency, online desalting effectiveness, and mass spectrometric data quality.

Methodology and Instrumentation

The workflow integrates multiple heart-cutting 2D-LC with online desalting:

• First-dimension separations:
    • IEX on Agilent Bio MAb NP5 column to resolve acidic, main, and basic charge variants
    • Protein A affinity capture on Agilent Bio-Monolith Protein A for mAb enrichment
• Second-dimension desalting:
    • Agilent AdvanceBio Desalting-RP cartridges remove nonvolatile salts online before MS
• Chromatographic gradients:
    • Salt gradient optimized with Agilent Buffer Advisor for IEX
    • Water/ACN gradient with 0.1% formic acid for RP desalting
• LC instrumentation:
    • Agilent 1290 Infinity 2D-LC system: quaternary and binary pumps, autosampler, dual thermostatted columns, multiport valves, and diode array detectors
• Mass spectrometry:
    • Agilent 6530 accurate-mass Q-TOF with ESI source operating at optimized gas temperatures, flows, and voltages
• Software:
    • Agilent OpenLAB CDS ChemStation, 2D-LC Acquisition Software, MassHunter Workstation, Buffer Advisor

Key Results and Discussion

• IEX → Desalting-RP:
    Innovator and biosimilar mAbs exhibit distinct acidic and basic variant profiles in the first dimension. Heart-cutting into the AdvanceBio Desalting-RP cartridge yields high-resolution MS spectra. Deconvoluted masses confirm variant identities and reveal C-terminal lysine truncation via 128 Da shifts.
• Affinity → Desalting-RP:
    Protein A capture isolates mAb1, mAb2, and mAb3; subsequent online desalting provides clean MS spectra, confirming expected glycoforms and sequence integrity without offline processing.
• Online desalting efficiently removes nonvolatile buffer components, enabling direct MS analysis, reducing sample handling and analysis time while maintaining sensitivity and spectral quality.

Benefits and Practical Applications

• Automated online desalting streamlines workflow and minimizes sample loss.
• Improved MS sensitivity and data quality for mAb charge variants and glycoforms.
• Applicable to QC laboratories, early-stage method development, and clone selection.
• Versatile configuration supports both charge variant and affinity-based separations.

Future Trends and Opportunities

Advancements in biotherapeutic analysis may include:

• Higher-throughput 2D-LC systems combining additional separation modes (e.g., HIC, reversed-phase) for multi-attribute workflows.
• Next-generation desalting materials offering lower backpressure and broader solvent compatibility.
• Integration of real-time data processing and machine learning for accelerated variant identification.
• Automated multi-attribute methods monitoring multiple post-translational modifications simultaneously.

Conclusion

Incorporating Agilent AdvanceBio Desalting-RP cartridges into a heart-cutting 2D-LC/MS platform effectively removes nonvolatile salts online and delivers high-quality mass spectrometric data for monoclonal antibody characterization. This approach simplifies workflows, enhances variant detection, and accelerates biotherapeutic development.

Reference

1. Buckenmaier S. Agilent 1290 Infinity 2D-LC Solution for Multiple Heart-Cutting. Agilent Technologies Application Note 5991-5615EN, 2015.
2. Krieger S. Application of Multiple Heart-Cutting 2D-LC in Method Development for Impurity Analysis – The Agilent 1290 Infinity 2D-LC Solution. Agilent Technologies Application Note 5991-5643EN, 2015.
3. Sandra K, Vanhoenacker G, Vandenheede I, Steenbeke M, Joseph M, Sandra P. Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine. Journal of Chromatography B, 2016.