Using Mass Detection as an Orthogonal Technology to Improve Routine Analysis of Biotherapeutics

Importance of the Topic

Biotherapeutics represent a rapidly expanding class of pharmaceutical products with global sales exceeding hundreds of billions of dollars. Routine quality control assays for identity, purity, and stability traditionally rely on HPLC-UV methods that may lack the specificity required for increasingly diverse and complex protein therapeutics. Integrating mass detection as an orthogonal technology enhances selectivity, sensitivity, and confidence in critical QC analyses.

Study Objectives and Overview

This application note demonstrates the use of a single-quadrupole mass detector (ACQUITY QDa) coupled with an LC-UV workflow to improve routine analysis of insulin analogues. Key objectives include:

• Developing MS-compatible chromatographic conditions for peptide mapping and intact protein analysis.
• Establishing a platform identity test capable of distinguishing multiple insulin analogues.
• Applying MS-assisted peak purity and stability assessments to monitor degradation products.

Methodology and Instrumentation

Peptide mapping and intact insulin analyses were performed on a Waters ACQUITY UPLC H-Class Bio system equipped with a TUV detector and QDa mass detector. Digested and intact samples were separated on a Peptide CSH C18 column using gradients optimized for both UV resolution and MS compatibility. Mobile phases contained 0.1% difluoroacetic acid to balance chromatographic selectivity and ionization efficiency. MS parameters included ESI+ mode, 350–1250 m/z range, 10 V cone voltage, and 500 °C probe temperature.

Main Results and Discussion

• Mobile Phase Optimization: Comparative evaluation of formic acid, TFA, and DFA showed that DFA provided baseline resolution of critical peptide pairs while maintaining strong MS signal-to-noise ratios.
• Identity Testing: A platform method using DFA enabled clear separation of digested insulin human, lispro, and glargine. MS data revealed mass shifts in unknown samples, allowing identification of insulin glulisine via characteristic +14.7 Da and +100.2 Da differences.
• Purity Assessment: Orthogonal MS detection permitted automated mass analysis across peak front, apex, and tail. Nearly identical spectra confirmed the absence of co-eluting impurities in intact insulin.
• Stability Monitoring: Time-course peptide maps of insulin human detected new peaks corresponding to deamidation (+1 Da) and pyroglutamate formation (+17 Da), illustrating the capacity to track degradation pathways.

Benefits and Practical Applications

Integrating the ACQUITY QDa into LC-UV workflows offers:

• Enhanced assay specificity and confidence by combining retention and mass information.
• High throughput analysis without extensive method redesign.
• Robust platform methods applicable across multiple analogues or biotherapeutic proteins.
• Improved detection of impurities and degradation products in QC and stability studies.

Future Trends and Applications

Continued advancements are expected in compact mass detectors, higher-resolution MS integration, and automated data processing. Broader adoption of orthogonal LC-UV/MS workflows will support regulatory modernization, facilitate biotherapeutic comparability studies, and enable real-time process monitoring in manufacturing.

Conclusion

This study highlights how the addition of a single-quadrupole mass detector to established LC-UV platforms significantly improves the specificity, sensitivity, and reliability of routine analyses for biotherapeutics. The demonstrated platform methods for insulin analogues can be readily adapted to other protein drugs, enhancing overall product understanding and quality assurance.

Used Instrumentation

• ACQUITY UPLC H-Class Bio System
• ACQUITY UPLC Tunable Ultraviolet (TUV) Detector
• ACQUITY QDa Mass Detector
• ACQUITY UPLC Peptide CSH C18 Column
• Empower 3 Chromatography Data Software

Reference

1. Evaluate Pharma, World Preview 2016, Outlook to 2022.
2. USP Monograph: Insulin Human [11061-68-0], Revision Bulletin 2015.
3. ICH Q10 Pharmaceutical Quality System, 2009.
4. Zhang X. et al., Waters Application Note 720005970EN, 2017.
5. Wagner B.M. et al., LCGC North America, 2015, 33(11):856–865.