INTACT Mass™ - a Versatile waters_connect™ Application for Rapid Mass Confirmation and Purity Assessment of Biotherapeutics

Significance of the Topic

The accurate determination of intact mass and impurity profiling of biotherapeutics is critical across discovery, process development, and quality control. High-throughput LC-MS methods coupled with automated data processing reduce cycle times and support compliance requirements in regulated environments.

Objectives and Study Overview

This application note describes the development and demonstration of the INTACT Mass™ software within the waters_connect™ informatics platform to streamline LC-MS intact mass confirmation and purity assessment. The workflow was tested on six commercial monoclonal antibodies and an IdeS-digested NISTmAb subunit standard to validate throughput, mass accuracy, and purity reporting.

Methodology and Instrumentation Used

System configuration:

• UPLC: ACQUITY™ Premier with binary solvent manager, FTN sample manager, and column heater.
• Column: ACQUITY Premier Protein BEH C4, 300 Å, 1.7 µm, 2.1 × 50 mm at 80 °C.
• Detectors: Tunable UV at 280 nm and ACQUITY RDa™ accurate mass detector (ESI positive, 400–7000 m/z).
• Software: waters_connect v2.1.2.4 with INTACT Mass application.

LC gradients: 2.5 min desalting run for intact mAbs; 3.0 min separation for subunits. MS settings included scheduled lockmass, 2 Hz scan rate, and high desolvation temperature.

Main Results and Discussion

High-throughput intact mAb screening (48 injections):

• Average mass error ~10 ppm across six mAbs with standard deviation <5 ppm.
• Real-time processing: up to five concurrent deconvolutions per sample.
• Dashboard flags enabled rapid pass/warning/fail status based on mass accuracy and purity thresholds.

Subunit analysis of NISTmAb:

• Separation of three subunits (scFc, LC, Fd) in 3 min with 99.1% combined peak purity.
• Average mass errors ~5 ppm for all subunits.

The automated MaxEnt1 and BayesSpray deconvolution algorithms self-optimize mass ranges and processing parameters, facilitating platform methods applicable to diverse biomolecule classes.

Benefits and Practical Applications of the Method

• Fully integrated acquire–process–report workflow in a compliance-ready environment.
• High throughput: 48 samples processed in ~136 min with combined acquisition and deconvolution.
• Flexible purity assessment via UV, TIC, or mass spectral data.
• Platform-based methods reduce method development effort for varied biomolecules.

Future Trends and Opportunities for Utilization

Expansion of the INTACT Mass application to other biomolecule classes, such as oligonucleotides and single-guide RNAs. Further integration with automated sample submission and advanced library searching for impurity and variant identification. Broader deployment in manufacturing quality control for real-time batch release testing.

Conclusion

The waters_connect INTACT Mass application delivers a streamlined LC-MS intact mass workflow, providing low-ppm mass confirmation and robust purity assessment. Its high throughput, automated deconvolution, and compliance features enable efficient screening of biotherapeutics from discovery to commercialization.

References

• Ferrige A. et al. Disentangling Electrospray Spectra with Maximum Entropy. Rapid Commun. Mass Spectrom. 1992;6:707–711.
• Skilling J. et al. Increasing Fidelity of Mass Spectral Deconvolution with Bayesian Probability. Waters Poster, 2010.
• Berger S., Boyce P. Beyond INTACT Mass Application within waters_connect. Waters Vblog, 2022.
• Doneanu C. et al. LC-MS Analysis of Single Guide RNA Impurities Using BioAccord and INTACT Mass. Waters Application Note, 2022.