Evaluating BioAccord System Performance in Late Stage Development and QC Environment – Peptide Mapping Analysis

Importance of the Topic

Liquid chromatography–mass spectrometry (LC-MS) peptide mapping plays a critical role in verifying protein primary sequences and monitoring product quality in biopharmaceutical development. Its reliability is essential for detecting post-translational modifications and ensuring batch-to-batch consistency under regulated conditions.

Study Objectives and Overview

This study aimed to demonstrate the reproducibility of the Waters BioAccord System for peptide mapping in late-stage development and QC environments. By conducting inter- and intra-system assessments using three identical platforms, the work addresses performance criteria required for traditional method validation.

Methodology

Using the Waters mAb Tryptic Digest Standard (NISTmAb), sample solutions were prepared at 0.2 µg/µL and 1 µg injected on column. Peptide separation was performed on an ACQUITY UPLC BEH C18 1.7 µm (2.1×100 mm) column at 65 °C with a 60-minute gradient from 99% to 60% aqueous mobile phase (0.1% formic acid) at 0.2 mL/min, followed by a total run time of 75 minutes including equilibration. Mass detection employed the ACQUITY RDa high-performance oa-TOF detector in positive ESI full-scan and fragmentation mode (2 Hz) over m/z 50–2000. Cone voltage was set to 30 V with a fragmentation cone ramp of 60–130 V, capillary at 1.5 kV, and desolvation at 500 °C. Data acquisition and processing were automated via waters_connect and the UNIFI Scientific Information System with a 10 ppm mass tolerance and minimum of three confirmatory fragments for peptide assignment.

Instrumentation

• ACQUITY UPLC I-Class PLUS System with optical detection (TUV/FLR)
• ACQUITY RDa Mass Detector (oa-TOF)
• waters_connect platform
• UNIFI Scientific Information System
• ACQUITY UPLC BEH C18 column, 1.7 µm, 2.1×100 mm

Main Results and Discussion

Overlay of total ion chromatograms from 18 injections over 22 hours revealed retention time standard deviations ≤0.09 minutes (5.4 seconds) across all monitored peptides. A set of native and modified peptides (including glycopeptides and oxidation/deamidation variants) showed an average relative abundance reproducibility (%RSD) within 8.12% across the three systems. These findings confirm both chromatographic stability and consistent MS response suitable for automated attribute monitoring.

Benefits and Practical Applications

The BioAccord System delivers robust, compliance-ready peptide mapping workflows with minimal manual intervention. Its reproducibility supports routine QC deployment and late-stage development efforts, facilitating reliable monitoring of critical quality attributes in biotherapeutics.

Future Trends and Opportunities

Advances in LC-MS automation, data analytics, and integration with information management systems will further streamline peptide mapping in regulated environments. Emerging techniques such as ion mobility separation and AI-driven data processing are poised to enhance throughput, sensitivity, and confidence in attribute monitoring applications.

Conclusion

Inter- and intra-system evaluations demonstrate that the BioAccord System meets stringent reproducibility requirements for peptide mapping in regulated workflows. Consistent retention times and modification quantification underscore its fitness for deployment in QC laboratories and late-stage biopharmaceutical development.

References

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