ADDING COST EFFECTIVE MASS DETECTION AS AN ORTHOGONAL TECHNIQUE FOR IMPROVED PRODUCTIVITY AND CONFIDENCE IN THE ANALYSIS OF PROTEIN BIOTHERAPEUTICS

Importance of the Topic

Peptide-based analyses are central to monitoring critical quality attributes (CQAs) in protein biotherapeutic development. While traditional optical detectors provide useful information on product identity, purity and potency, they lack selectivity in complex mixtures and can struggle with co-eluting species and low-abundance variants. Integrating mass detection as an orthogonal technique boosts specificity, sensitivity and confidence in routine assays, ultimately enhancing data reliability and streamlining workflows.

Objectives and Study Overview

The study aimed to:

1. Evaluate the performance of the ACQUITY QDa mass detector in a peptide mapping workflow for a monoclonal antibody (trastuzumab).
2. Compare peptide profiling under different ion-pairing conditions (TFA vs. FA).
3. Demonstrate the benefits of selected ion recording (SIR) in resolving co-eluting and low-abundance species.
4. Extend the approach to glycopeptide profiling, released glycan analysis and identity screening of complementarity-determining region (CDR) peptides.

Methods and Instrumentation

A tryptic digest of trastuzumab (4 μg on column) was analyzed using an ACQUITY UPLC H-Class system with TUV detection and ACQUITY QDa. Two reversed-phase columns (CSH C18 and BEH C18) were tested with mobile phases containing either 0.1% TFA or 0.1% FA. The QDa was operated in positive mode (mass range 350–1250 Da, cone voltage 10 V, capillary 1.5 kV, probe 500 °C) at 2 Hz acquisition, with SIR for targeted peptides and glycoforms.

Instrumentation Used

• ACQUITY UPLC H-Class system
• ACQUITY UPLC TUV detector (215 nm)
• ACQUITY QDa mass detector
• CSH C18, 2.1×100 mm, 1.7 μm column
• BEH C18, 2.1×150 mm, 1.7 μm column

Key Results and Discussion

• Mass accuracy remained within ±0.3 Da across a broad range of peptide masses, achieving ~90% sequence coverage.
• Peptide profiles were consistent using TFA or FA; the QDa delivered reliable spectral data under both ion-pairing conditions.
• SIR improved quantification of co-eluting isobaric peptides (e.g. T26 vs. T10), reducing quantitation errors seen by UV alone.
• Low-abundance variants, such as oxidized T21 and deamidated CDR peptides, were clearly identified and quantified with high specificity.
• Glycopeptide monitoring and released glycan analysis (30 min RapiFluor-MS workflow) showcased efficient detection of key N-glycoforms (e.g. G0F, G1F, G2F).
• CDR peptide profiling enabled rapid identity confirmation and monitoring of critical modifications affecting binding efficiency.

Benefits and Practical Applications

• Enhanced specificity for complex peptide mixtures through orthogonal mass detection.
• Improved sensitivity toward low-abundance species and post-translational modifications.
• Streamlined workflows by combining UV and MS detectors in a single platform.
• Actionable data for QA/QC in biotherapeutic production, including identity, purity and CQA monitoring.
• Compatibility with existing LC methods and ion-pairing reagents minimizing method redevelopments.

Future Trends and Opportunities

As biotherapeutic complexity grows, integrated, cost-effective mass detectors like the QDa will play a larger role in multi-attribute monitoring. Advances in automation, data processing algorithms and expanded mass range capability could further enhance throughput and analytical depth. Future developments may include real-time process monitoring, more sophisticated glycoform quantitation and integration with AI-driven data analytics for predictive quality control.

Conclusion

The ACQUITY QDa provides a fit-for-purpose, orthogonal mass detection technique that complements UV-based peptide assays, offering improved specificity, sensitivity and confidence in biotherapeutic analysis. Its compact design and compatibility with standard LC workflows enable seamless integration into routine QA/QC laboratories, ultimately boosting productivity and data integrity.

References

No external references were provided in the source text.