Monitoring Multiple Attributes in a Single Assay Using the ACQUITY QDa Detector for Product Confirmation and Process Monitoring of Product Quality Attributes

Importance of the Topic

The monitoring of multiple product quality attributes (PQAs) in a single assay is essential for modern biopharmaceutical development and quality control. A unified LC-MS method streamlines analysis, reduces sample requirements, and supports Quality by Design (QbD) principles by delivering detailed molecular information on critical quality attributes.

Study Objectives and Overview

This study demonstrates a proof-of-concept workflow using the Waters ACQUITY QDa Detector coupled with Empower software to perform both product confirmation and routine screening of post-translational modifications in a single acquisition. Trastuzumab was used as a model monoclonal antibody, and key attributes including complementarity determining region (CDR) peptides, oxidized and deamidated peptides, and glycopeptides were targeted.

Methodology and Instrumentation

A tryptic digest of reduced and alkylated trastuzumab was analyzed on an ACQUITY UPLC H-Class Bio System with the following setup:

• Column: ACQUITY UPLC Peptide CSH C18, 1.7 µm, 2.1×100 mm, 65 °C
• Detectors: ACQUITY UPLC Tunable UV (215 nm) and ACQUITY QDa Detector (ESI+, 350–1250 Da)
• Mobile phases: water +0.1% formic acid (A), acetonitrile +0.1% formic acid (B)
• Gradient: 97% A to 20% A over 131 min, flow 0.2 mL/min
• Data system: Empower 3 CDS

Extracted ion chromatograms (XICs) were generated using derived channels for multiple m/z values of interest. Selected Ion Recording (SIR) was applied for enhanced sensitivity in glycopeptide quantitation.

Main Results and Discussion

Derived channels allowed simultaneous extraction of six CDR peptides, confirming trastuzumab identity by retention time and mass. Oxidized and deamidated forms of specific peptides were monitored by switching target m/z values at defined retention windows to avoid signal overlap. Overlay of five SIR channels enabled accurate relative quantitation of major glycoforms (G0F, G1F, G2F, G0, Man5). Empower processing methods automated peak integration and custom calculations to report relative abundances and confirm compliance.

Benefits and Practical Applications

This multi-attribute monitoring approach reduces the need for separate optical assays, increases molecular specificity, and cuts overall analysis time. The use of ACQUITY QDa provides a cost-effective MS solution for routine QC and process monitoring, aligning with regulatory expectations for QbD and advanced product characterization.

Future Trends and Opportunities

As biopharmaceutical complexity grows, expanding multi-attribute methods to include additional modifications and product variants will be critical. Integration with high-throughput automation and advanced data analytics will further enhance process control and accelerate method adoption in regulated environments.

Conclusion

The ACQUITY QDa Detector, together with Empower software, offers an effective single-assay strategy for monitoring identity and multiple PQAs. This workflow demonstrates reliable detection and quantitation of CDR peptides, chemical modifications, and glycoforms, supporting its implementation for product confirmation and quality monitoring.

Reference

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