Analysis of Monoclonal Antibody (mAb) Using Agilent 1290 Infinity LC System Coupled to Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF)

Importance of the topic

The global demand for monoclonal antibodies (mAbs) continues to grow rapidly, driven by their use in therapeutic and diagnostic applications. High-throughput and high-resolution analytical techniques are essential to ensure product quality, monitor glycosylation microheterogeneity, and support comparability and biosimilar studies.

Objectives and overview

This study demonstrates a robust liquid chromatography–mass spectrometry (LC-MS) approach to analyze intact and reduced forms of two IgG subclasses (IgG1 and IgG2). The goals include achieving sharp chromatographic peaks, accurate mass profiles, and rapid visual comparison using mirror plots.

Methodology

Samples of IgG1 and IgG2 were diluted to 100 µg/mL in 0.1 percent formic acid. Intact mAbs were directly injected (1 µL, 100 ng), while reduction was performed with dithiothreitol at 60 °C for 1 hour to generate heavy and light chains. A segmented gradient with isopropanol, acetonitrile, water, and formic acid was applied over a 10-minute run, followed by re-equilibration.

Used Instrumentation

• Agilent 1290 Infinity LC System with binary pump
• Agilent Poroshell 300SB-C8 column (2.1×75 mm, 5 µm)
• Agilent 6530 Accurate-Mass Q-TOF with JetStream ESI source
• Agilent MassHunter Qualitative Analysis and BioConfirm softwares with pMod deconvolution algorithm

Main results and discussion

Chromatograms showed narrow peaks (~0.5 min at base) for intact mAbs, and clear separation of light and heavy chains in reduced samples. Deconvoluted masses revealed distinct major peaks at 144 944 Da for IgG1 and 144 498 Da for IgG2, reflecting five versus four dominant glycoforms, respectively. Light chains were identical (~22 929 Da) for both antibodies, while heavy chains exhibited subclass-specific mass distributions: IgG1 from 49 475 to 49 799 Da and IgG2 from 49 255 to 49 581 Da. Mirror plots enabled direct comparison and highlighted differences localized to the heavy chain, with satellite peaks indicating salt adducts.

Benefits and practical applications

• Rapid quality control and impurity profiling of mAbs
• Efficient comparability assessments for originator and biosimilar products
• High mass accuracy for glycoform and subunit characterization
• Streamlined data analysis with automated deconvolution and mirror plotting

Future trends and potential applications

Advances in multi-attribute methods will integrate glycan, oxidation, and clipping analyses into a single workflow. Automation of sample preparation and data interpretation, coupled with machine-learning algorithms, will further accelerate mAb characterization. Native MS and orthogonal separations such as hydrophilic interaction chromatography (HILIC) promise deeper insight into higher-order structure and glycoform diversity.

Conclusion

The described Agilent 1290 Infinity LC–6530 Q-TOF platform delivers fast, high-resolution analysis of intact and reduced mAbs. Combined with MassHunter BioConfirm software, it provides robust mass accuracy, clear glycoform profiling, and powerful mirror-plot comparisons, making it well suited for routine QC, comparability, and biosimilar development studies.

Reference

• World Preview 2013, Outlook to 2018, EvaluatePharma Executive Summary, 2013.
• Agilent pMod Deconvolution Algorithm, publication 5991-2225EN.