Critical Quality Attribute Monitoring of mAbs at the Intact and Subunit Levels Using a Cost-Effective, Simple and Robust LC/MS Solution

Importance of the Topic

Monitoring critical quality attributes (CQAs) such as molecular mass and glycosylation profiles of monoclonal antibodies (mAbs) is essential for ensuring drug safety, efficacy, and batch-to-batch consistency in biopharmaceutical production.

Objectives and Overview of the Study

This application note evaluates a cost-effective single quadrupole LC/MS solution (Agilent InfinityLab LC/MSD XT) for intact and subunit-level mAb analysis. Two antibodies (NISTmAb and a model IgG1, mAb1) were processed by four workflows—dilution, deglycosylation, IdeS digestion, and reduction—to generate samples spanning 23–148 kDa.

Methodology and Instrumentation

An Agilent 1290 Infinity II LC system coupled to an Agilent LC/MSD XT mass selective detector (10–3000 m/z, Jet Stream source) was used. Separations employed a PLRP-S column (2.1 × 50 mm, 5 µm, 1000 Å) at 80 °C with a water/acetonitrile gradient (0.1% formic acid). Data acquisition and deconvolution utilized Agilent OpenLab ChemStation software.

Main Results and Discussion

The LC/MSD XT delivered accurate mass measurements across all sample types. Intact mAbs (~148 kDa) yielded charge envelopes between 2000–3000 m/z, resolving five major glycoforms with mass errors below 136 ppm. PNGase F-deglycosylated mAbs collapsed to single ~145 kDa peaks (mass errors <52 ppm). IdeS-generated Fc/2 and F(ab’)2 subunits (25 and 98 kDa) and reduced light/heavy chains (23–51 kDa) were tracked with mass errors within ±64 ppm. This underscores the method’s robustness for QC environments.

Benefits and Practical Applications of the Method

• Cost-effective single quadrupole instrument reduces operational expenses.
• Robust and user-friendly workflow suited for QC laboratories.
• Rapid sample preparation strategies covering intact, deglycosylated, digested, and reduced forms.
• Accurate mass determination supports glycoform profiling and CQA monitoring.

Future Trends and Opportunities

Emerging developments may include integration of higher-sensitivity detectors, automated sample preparation, expansion into multi-attribute method (MAM) workflows, and coupling with advanced separation media for enhanced resolution. Real-time process analytics and hybrid instrumentation could further accelerate biopharma quality control.

Conclusion

The Agilent InfinityLab LC/MSD XT offers a simple, rapid, and reliable solution for intact and subunit mass analysis of mAbs in QC settings. Its broad mass range, robust operation, and deconvolution capabilities enable effective CQA monitoring across diverse sample types.

References

• Liu P. et al. Rapid Commun Mass Spectrom. 2019, 33(1), 31–40.
• OpenLab CDS ChemStation Edition with Content Management Systems User's Guide. Agilent Technologies, M8301-90089.
• OpenLab CDS ChemStation Edition C.01.09 Administrator Guide. Agilent Technologies, M8305-90019.
• Kilpatrick EL et al. Protein Expression Purif. 2012, 85, 94–99.
• Profiling Glycosylation of mAbs Using the Agilent 6545XT AdvanceBio LC/Q-TOF. Agilent Technologies, 5991-8796EN.