Adding Mass Detection to Routine Peptide-Level Biotherapeutic Analyses with the ACQUITY QDa Detector

Importance of Topic

The addition of mass detection to routine peptide‐level analysis addresses a critical need in biotherapeutic development and quality control. Peptide mapping provides detailed information on protein identity, post‐translational modifications, and purity. Integrating an affordable, compact mass detector alongside traditional optical detectors enhances data confidence, accelerates method development, and reinforces compliance in regulated environments.

Objectives and Study Overview

This application note evaluates the Waters ACQUITY QDa single‐quadrupole mass detector as an orthogonal addition to existing LC–UV peptide assays. The goals are to demonstrate:

• Compatibility of the QDa detector with legacy mobile phases containing trifluoroacetic acid (TFA).
• Peptide coverage and detection across a broad molecular weight range typical of biotherapeutic digests.
• Performance improvements when using formic acid (FA) and novel stationary phases.

Methodology and Instrumentation

A tryptic digest of the therapeutic monoclonal antibody trastuzumab was prepared at 0.5 mg/mL. UPLC separations employed an ACQUITY UPLC H-Class system with inline TUV and QDa detectors and either a BEH 300 Å C18 column (2.1 × 100 mm, 1.7 µm) using 0.1% TFA mobile phases or a Peptide CSH 130 Å C18 column with 0.1% FA mobile phases. A 120-minute gradient at 0.2 mL/min was used, with MS settings of 350–1250 Da mass range, 10 V cone voltage, and 1.5 kV capillary voltage.

Main Results and Discussion

Simultaneous UV and MS peptide maps showed a high degree of correlation, confirming QDa’s compatibility with TFA‐based methods. Switching to FA and a CSH column improved MS signal intensity. A charge‐state analysis of heavy‐chain peptides revealed that over 90% of expected fragments were detected with multiple charge states using either TFA or FA. Mass accuracy for the base peak ions remained within ±0.2 Da, meeting instrument specifications and ensuring reliable mass assignments.

Benefits and Practical Applications

The ACQUITY QDa detector provides:

• A cost-effective orthogonal detection method requiring minimal changes to existing workflows.
• Enhanced confidence in peptide identification and purity assessments for biotherapeutic QC.
• Flexibility to employ legacy ion-pairing chemistries or transition to MS-friendly FA conditions and advanced columns.

Future Trends and Possibilities

Emerging developments may include:

• Integration of higher‐resolution mass detectors for more detailed multi‐attribute methods.
• Automation and real-time data processing within GMP‐compliant software environments.
• Broader application to larger peptides and intact protein analysis in a routine QC setting.

Conclusion

The ACQUITY QDa detector effectively supplements routine LC–UV peptide assays by delivering accurate mass data across a wide mass range. Its compatibility with both TFA and FA mobile phases and easy integration into existing UPLC systems make it an ideal choice for biopharmaceutical peptide mapping and quality assurance.

References

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