Native SEC/MS Analysis of mAbs on an Entirely Biocompatible Flow Path

Significance of Topic

Size exclusion chromatography coupled to native mass spectrometry is critical for monitoring monoclonal antibody size variants that impact safety and efficacy. Eliminating desalting steps accelerates workflows and preserves protein native structure, enabling accurate quality control and structural analysis.

Objectives and Study Overview

This work assesses the performance of a fully biocompatible Agilent 1290 Infinity II Bio LC System combined with a PEEK-lined AdvanceBio SEC column using volatile ammonium acetate buffers for direct native SEC–MS analysis of mAbs. The study compares this setup to traditional stainless steel hardware with phosphate and acetate mobile phases.

Methodology and Instrumentation

Size exclusion separations were performed under non denaturing aqueous conditions with 150 mM phosphate and 50–100 mM ammonium acetate at pH 7. Columns evaluated include stainless steel and PEEK-lined AdvanceBio SEC 200 Å, 1.9 µm. Systems tested are the standard 1290 Infinity II LC and the Bio LC with iron-free MP35N flow path. Detection used UV at 280 nm and native electrospray ionization on an Agilent 6545 LC/Q-TOF. Key parameters covered flow rate, injection volume, temperature, and MS source settings for high resolution native analysis.

Main Results and Discussion

• Phosphate mobile phases deliver consistent separation and HMW recovery across all hardware configurations.
• Switching to low ionic strength ammonium acetate on stainless steel hardware causes peak broadening, tailing, and loss of high molecular weight species due to nonspecific surface interactions.
• The fully biocompatible Bio LC system with a PEEK-lined column restores sharp peaks, preserves aggregates, and supports direct native SEC–MS coupling without additional desalting.
• Native SEC–MS of NISTmAb under these conditions resolves HMW aggregates, monomer glycoforms, and low molecular weight fragments in one run.
• Analysis of the ADC brentuximab vedotin under native conditions reveals noncovalent light and heavy chain assemblies, drug distribution from DAR0 to DAR8, average DAR of 4, and glycosylation variants.

Benefits and Practical Applications

This biocompatible flow path and volatile buffer strategy streamlines mAb and ADC characterization workflows by removing offline desalting and two-dimensional setups. It improves throughput in QC labs, enables detailed critical quality attribute monitoring, and maintains protein native conformation for more accurate mass and structural assignments.

Future Trends and Opportunities

Advances in inert column materials and integration with high resolution MS will expand direct native SEC–MS applications to complex biotherapeutics. Developments in automation, alternative buffer chemistries, and novel column formats will enhance sensitivity, robustness, and routine adoption in pharmaceutical research and production environments.

Conclusion

Adoption of fully biocompatible flow paths combined with volatile, MS-compatible mobile phases enables seamless native SEC–MS analysis of monoclonal antibodies and antibody–drug conjugates. This approach reduces complexity, preserves native structures, and delivers comprehensive characterization of aggregates, fragments, glycoforms, and drug conjugation profiles in a single analytical run.

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