Demonstrate Method Robustness and Seamless Method Transfer, a Step Towards Enabling High Resolution Accurate Mass Multi-Attribute Method for Biotherapeutic QC

Importance of the Topic

High-resolution accurate mass (HRAM) peptide mapping is a cornerstone in biotherapeutic characterization, enabling simultaneous identification, quantitation and monitoring of product quality attributes (PQAs) and trace impurities. Robust performance evaluation and seamless method transfer are critical for deploying HRAM multi-attribute methods (MAM) in regulated quality control environments.

Objectives and Study Overview

This work aimed to develop and validate a system performance evaluation test (SET) based on a pre-digested bovine serum albumin (BSA) standard, integrated within a Chromeleon eWorkflow, to:

• Define acceptance criteria for key LC-MS performance attributes.
• Demonstrate reproducible peptide mapping across Orbitrap Exploris 240 and MX platforms.
• Enable method transfer and consistent PQA analysis of a NISTmAb digest across instruments and sites.

Methodology

A pre-digested BSA sample (1 pmol/µL) was used in a defined injection sequence to monitor mass accuracy, isotopic fidelity, retention time, peak area, height, width and modification levels for a panel of five signature peptides. Once the SET passed predefined criteria, the same eWorkflow was applied to analyze a NISTmAb digest, assessing multiple PQAs and new peak detection.

Used Instrumentation

• Thermo Scientific Vanquish UHPLC systems (Horizon or Flex)
• Hypersil GOLD Vanquish C18 column (150 × 2.1 mm, 1.9 μm)
• Thermo Scientific Orbitrap Exploris 240 and Exploris MX mass spectrometers
• Chromeleon 7.3.1 Chromatography Data System with eWorkflows

Key Results and Discussion

SET performance on both Exploris platforms consistently met acceptance criteria:

• Mass accuracy <3 ppm for top three confirming ions
• Retention time RSD <2%
• Peak area RSD <10%
• Isotopic dot product ≥0.9

Cross-platform evaluation of NISTmAb PQAs demonstrated:

• Intra-instrument repeatability across 10 replicates: PQA %CV <5%
• Inter-instrument variability: %CV well below 5% for glycosylation, oxidation, deamidation and other attributes
• New peak detection algorithm in Chromeleon reliably identified spiked PRTC peptides without false positives.

Benefits and Practical Applications

This approach delivers a unified workflow for installation testing, routine QC and troubleshooting of HRAM MAM methods. The SET ensures consistent instrument performance, while eWorkflows guarantee method reproducibility and straightforward transfer between labs and platforms.

Future Trends and Possibilities

Continued developments may include:

• Extension to a broader range of biotherapeutic formats and complex matrices
• Real-time system performance monitoring and feedback loops
• Machine-learning-driven acceptance criteria and anomaly detection
• Integration with regulatory compliance frameworks for accelerated method validation

Conclusion

A comprehensive SET based on a BSA digest and implemented in Chromeleon eWorkflows provides a robust foundation for HRAM MAM deployment in biotherapeutic QC. Both Orbitrap Exploris 240 and MX systems exhibited excellent performance, method transferability and reproducible PQA quantitation across platforms.

References

1. Thermo Fisher Scientific, “A high-resolution accurate mass multi-attribute method for critical quality attribute monitoring and new peak detection”, Application Note 72916