ADDING COST EFFECTIVE MASS DETECTION AS AN ORTHOGONALTECHNIQUE FOR IMPROVED PRODUCTIVITY AND CONFIDENCEIN THE ANALYSIS OF PROTEIN BIOTHERAPEUTICS

Importance of the Topic

Biotherapeutic development relies on accurate monitoring of critical quality attributes (CQAs) during peptide mapping workflows. While UV detection provides initial assurance of purity and identity, mass detection offers an orthogonal approach that enhances specificity, lowers detection limits, and increases confidence in data interpretation.

Objectives and Study Overview

This study demonstrates a unified workflow combining UV absorbance and inline mass spectrometry using the Waters ACQUITY QDa detector integrated with UPLC. The approach was applied to a trypsin digest of trastuzumab to assess product identity, homogeneity, and low-abundance variants in a single cost-effective assay.

Methodology

A monoclonal antibody (trastuzumab) digest was separated by reversed-phase liquid chromatography using two ion-pairing conditions (0.1% TFA or 0.1% formic acid). A binary gradient (water/acid and acetonitrile/acid) at 0.2 mL/min on CSH and BEH300 C18 columns (2.1×100 mm, 1.7 µm) at 65 °C was employed. UV absorbance at 215 nm and in-line mass detection (selected ion recording, full scan) were collected to monitor peptide masses, post-translational modifications, and glycopeptide profiles.

Used Instrumentation

• ACQUITY UPLC H-Class system
• ACQUITY UPLC TUV detector (215 nm)
• ACQUITY QDa single quadrupole mass detector
• Columns: CSH C18 and BEH300 C18, 2.1×100 mm, 1.7 µm
• Mass range: 350–1250 Da; cone voltage: 10 V; capillary voltage: 1.5 kV; probe temperature: 500 °C

Main Results and Discussion

The ACQUITY QDa demonstrated fit-for-purpose performance with both TFA and formic acid mobile phases, delivering robust mass spectra across a broad molecular weight range.

• Specificity: Selected ion recording (SIR) resolved co-eluting peptides, enabling accurate quantitation of species such as T10 and T26.
• Sensitivity: Low-abundance oxidized peptides (e.g., T21-oxidized) were clearly detected with minimized baseline noise.
• Identity Screening: Complementarity-determining region (CDR) peptides were selectively extracted from full scans for rapid confirmation of drug identity.
• PTM Monitoring: Deamidated forms of CDR peptides were quantified despite co-elution, enhancing quality assurance.
• Glycan Analysis: Inline SIR enabled targeted monitoring of glycopeptides under RPLC, and released glycans labeled for HILIC-FLR/QDa provided mass confirmation in under 30 minutes.
• Mass Accuracy: Observed mass errors remained within ±0.3 Da across peptides up to 8 kDa.

Benefits and Practical Applications

The combined UV/MS workflow offers:

• Orthogonal confirmation of peptide identity and purity.
• Compatibility with existing UV-based assays using TFA or formic acid.
• Enhanced throughput and reduced development timelines.
• Selective quantification of variants and low-abundance species impacting efficacy.
• Streamlined peptide mapping, PTM, and glycan profiling in a single system.

Future Trends and Potential Applications

Emerging applications may include:

• Routine inline mass detection across diverse biotherapeutic molecules.
• Quantitative assays incorporating stable-isotope standards.
• Automated data processing for rapid CQA reporting.
• Real-time process analytical technology (PAT) for continuous monitoring.

Conclusion

The ACQUITY QDa detector provides a cost-effective, easy-to-use orthogonal mass detection platform that integrates seamlessly with UPLC-UV workflows. It strengthens confidence in peptide mapping assays by delivering specific, sensitive, and accurate monitoring of critical quality attributes in biotherapeutic development environments.