The MAM 2.0 workflow enables seamless transition from research and development to quality control

Significance of the Topic

Multi‐attribute method (MAM) workflows based on high‐resolution accurate‐mass LC‐MS are emerging as key tools in biopharmaceutical development and quality control. They enable direct, simultaneous monitoring of multiple critical product quality attributes (PQAs), supporting risk‐based and quality‐by‐design strategies. Seamless transfer of MAM assays from research to regulated QC environments addresses industry challenges related to reproducibility, instrument variation, and compliance, ultimately accelerating biotherapeutic development and lifecycle management.

Goals and Overview

This study demonstrates an end‐to‐end MAM 2.0 workflow that enables effortless method transfer from a research setting into a QC environment. Key objectives included:

• Developing a robust MAM assay on an Orbitrap Exploris 240 mass spectrometer coupled to UHPLC
• Packaging acquisition, processing, and reporting components into a Chromeleon eWorkflow procedure
• Validating seamless deployment onto two Orbitrap Exploris MX detectors with minimal reconfiguration
• Evaluating reproducibility and quantitative comparability across instruments

Methodology and Instrumentation

A recombinant monoclonal antibody standard (NISTmAb) was digested with trypsin and aliquoted for analysis. Peptides were separated on a Hypersil GOLD VANQUISH C18 column using a Vanquish Horizon or Vanquish Flex UHPLC system with a water/acetonitrile gradient containing 0.1% formic acid. Data‐dependent MS2 (Top 5 ddMS2) on the Orbitrap Exploris 240 identified key PQAs—deamidation, oxidation, glycation, isomerization, succinimide formation, glycosylation, and C‐terminal lysine clipping. A targeted full‐MS assay was then defined for quantitative monitoring. Chromeleon 7.3.1 CDS managed acquisition and processing, and BioPharma Finder 5.0 was used for peptide mapping and target list generation. A single eWorkflow package contained all LC and MS methods, peak‐integration parameters, pass/fail criteria, and report templates.

Instrumentation Used

• Thermo Scientific™ Orbitrap Exploris 240 mass spectrometer
• Thermo Scientific™ Orbitrap Exploris MX mass detector
• Thermo Scientific™ Vanquish™ Horizon UHPLC system
• Thermo Scientific™ Vanquish™ Flex UHPLC system
• Thermo Scientific™ Chromeleon™ CDS Enterprise with eWorkflow
• Thermo Scientific™ BioPharma Finder™ software v5.0

Main Results and Discussion

Using the eWorkflow procedure, the MAM assay was deployed in parallel on one Exploris 240 and two Exploris MX instruments connected to the same network. Ten replicate injections of NISTmAb digest on each system yielded highly comparable quantitative results for all monitored PQAs. Instrument‐to‐instrument relative standard deviations remained below 10%, even for low‐abundance glycoforms (<1% relative level). The Exploris MX detector achieved better than 5% RSD for oxidation, deamidation, glycation, and isomerization metrics, demonstrating excellent precision and stability. A one‐point internal mass calibration feature on the Exploris MX maintained sub‐3 ppm mass accuracy over weeks without full recalibration. Automated pass/fail reports provided visual summaries and electronic signatures for compliance.

Benefits and Practical Applications

• Eliminates manual method re‐development when migrating from R&D to QC
• Ensures consistency and reproducibility across multiple instruments and sites
• Integrates data acquisition, processing, and reporting into a single regulated workflow
• Reduces instrument‐to‐instrument variation, meeting global QC deployment requirements
• Supports rapid decision‐making by providing automated pass/fail evaluation of PQAs

Future Trends and Opportunities

• Expansion of MAM workflows to additional biotherapeutic formats (e.g., bispecifics, ADCs)
• Integration with laboratory information management systems for end‐to‐end data traceability
• Advanced data analytics and machine learning for deeper PTM profiling and degradation prediction
• Broader adoption of compliance‐ready, networked eWorkflows across global QC labs
• Further simplification of instrument maintenance via embedded calibration and self‐diagnostics

Conclusion

The MAM 2.0 eWorkflow demonstrated seamless transfer of an NISTmAb peptide mapping and PQA assay from research (Orbitrap Exploris 240) to QC (Orbitrap Exploris MX) with minimal method changes. Consistent, high‐precision quantitation of critical quality attributes was achieved across multiple instruments, underscoring the robustness and compliance readiness of the approach. This unified workflow paves the way for widespread adoption of MAM in regulated environments, enhancing productivity and ensuring product quality.

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