LC/MS of Intact Therapeutic Monoclonal Antibodies Using Agilent AdvanceBio RP-mAb

Importance of the Topic

Monoclonal antibodies (mAbs) have become essential in modern biopharmaceutical research and therapeutics. Post-translational modifications generate microheterogeneity, making sensitive monitoring of glycosylation and conjugation critical to ensure consistent drug safety and efficacy.

Objectives and Study Overview

This application note evaluates Agilent AdvanceBio RP-mAb columns for rapid, high-resolution LC/MS analysis of intact therapeutic mAbs (mAb1, mAb2) and a lysine-conjugated antibody-drug conjugate (mAb3). Key aims were to assess chromatographic speed, reproducibility, and accurate mass determination for glycoform and drug-load profiling.

Methodology and Instrumentation

Samples were prepared at 1 µg/µL in 0.1% formic acid/3% acetonitrile with 1 µL injections. Instrumentation Used:

• LC: Agilent 1290 Infinity with AdvanceBio RP-mAb C4 (2.1×50 mm) and Diphenyl (2.1×75 mm) columns, superficially porous 3.5 µm, 450 Å pores.
• Mobile phases: A = 0.1% formic acid in water; B = 80% isopropanol:10% acetonitrile:9.9% water plus 0.1% formic acid. Gradient from 20% to 90% B over 11.1 min at 0.6 mL/min; column heated to 80 °C.
• MS: Agilent 6530 Accurate-Mass Q-TOF with dual AJS ESI in positive mode, m/z range 2,000–6,000, data at 1 GHz. Deconvolution via maximum entropy and pMod in MassHunter BioConfirm.

Main Results and Discussion

Both C4 and Diphenyl columns produced narrow total ion chromatogram peaks (~0.5 min at base) with high signal-to-noise. Deconvoluted spectra identified five major glycoforms for mAb1 and four for mAb2. The ADC mAb3 displayed eight discrete drug-load species (D0–D8) with clear mass increments corresponding to each payload addition.

Benefits and Practical Applications

The superficially porous particle design shortens diffusion paths, enabling high linear velocities and efficient separations. This approach accelerates throughput for batch-to-batch consistency studies and delivers precise profiling of glycosylation patterns and conjugation levels for quality control.

Future Trends and Applications

Emerging directions include coupling with native MS for structural analysis, microflow and nanoLC adaptations to reduce solvent consumption, fully automated high-throughput workflows, and exploration of novel chemistries to further enhance selectivity and resolution.

Conclusion

Agilent AdvanceBio RP-mAb columns paired with an Accurate-Mass Q-TOF offer fast, reproducible, and high-resolution analysis of intact mAbs and ADCs, empowering detailed mass profiling of glycoforms and drug-load distributions.

Reference

1. Gritti F. Chromatography Today June 2012, 4–11.
2. Gudihal R, Suresh Babu CV, Tang N. Monoclonal Antibody Analysis Using Agilent 1290 LC and 6530 Accurate-Mass Q-TOF; Agilent Application Note 5991-4266EN, 2014.