Deploying the multi-attribute method (MAM) across sites at Pfizer

Significance of the Topic

MAM (Multi-Attribute Method) integrates liquid chromatography–mass spectrometry (LC-MS) peptide mapping and automation to simultaneously monitor multiple product quality attributes (PQAs) of biotherapeutics with site specificity. By consolidating routine profile‐based assays into a single platform, MAM enhances process and product understanding, accelerates development timelines, and supports Quality by Design initiatives in pharmaceutical R&D.

Goals and Overview

Pfizer’s Biotherapeutics Pharmaceutical Sciences (BioTx PS) group aims to deploy MAM across global sites to streamline analytical workflows from early discovery through commercialization. The objectives include:

• Implementing a “lab‐of‐the‐future” MAM platform for consistent PQA monitoring.
• Replacing multiple release, identity and stability assays with a unified LC-MS method.
• Enabling real‐time process development decisions by providing direct quantitative and site‐specific PQA data.

Methodology and Instrumentation

The MAM workflow begins with rapid tryptic digestion to preserve labile posttranslational modifications and minimize artificial artifacts. Initial in-depth PQA characterization is performed by LC-MS/MS (CID/HCD/ETD) using high-resolution Orbitrap mass spectrometers to build a targeted peptide workbook. Subsequently, routine monitoring is conducted in full-MS mode on Exactive Plus systems controlled by Chromeleon™ CDS, leveraging chromatographic retention time, accurate mass and isotope patterns for automated PQA quantitation. System suitability is managed with an in-house mAb standard and the Pierce BSA Protein Digest, ensuring consistent UHPLC and MS performance.

Main Results and Discussion

Comparisons of MAM with conventional assays demonstrated excellent agreement: N-glycoform profiles matched established 2-AB labelled glycan methods for major, minor and trace species across multiple bioreactor samples. In an 18-week thermal stress study, MAM accurately resolved and quantified charge variants (e.g., deamidation, sialylation) at specific sites, surpassing global techniques like imaged capillary electrophoresis by providing molecular identification and localization. Site‐specific lysine glycation trends revealed formulation effects, and non-targeted analysis detected unexpected Fc clipping, which was back-characterized by MS/MS and incorporated into the PQA library.

Benefits and Practical Applications

MAM offers:

• Consolidation of multiple assays into one LC-MS workflow, reducing time and resource requirements.
• Direct, quantitative, site-specific monitoring of known and novel PQAs.
• Automated data processing and reporting, minimizing user intervention.
• Retrospective data mining and trend analysis enabled by Chromeleon CDS and enterprise servers.

Future Trends and Opportunities

Wider adoption of MAM in non-GMP and QC labs will depend on further instrument simplification, robustness and user-friendly software. Increased automation, reduced footprint hardware and turnkey eWorkflows are expected to lower the barrier for non-expert users. Integration into GMP environments is targeted within 3–5 years as regulatory frameworks evolve and MAM platforms mature.

Conclusion

Pfizer’s deployment of a multi‐site MAM lab-of-the-future in collaboration with Thermo Fisher Scientific demonstrates that a single LC-MS method can robustly replace multiple traditional assays, providing enhanced product quality insights and supporting accelerated biotherapeutic development.

Instrumentation Used

• Thermo Scientific Q Exactive Plus and Exactive Plus Hybrid Quadrupole‐Orbitrap mass spectrometers
• Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer
• Thermo Scientific Vanquish and Vanquish Duo UHPLC systems
• Thermo Scientific Chromeleon Chromatography Data System
• Thermo Scientific BioPharma Finder software
• Thermo Scientific Pierce BSA Protein Digest and in-house mAb standards for system suitability

References

• Rogers RS, Nightlinger NS, Livingston B, Campbell P, Bailey R, Balland A. Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics. mAbs. 2015;7(5):881–890.
• Andrew Dawdy. Understanding Biotherapeutic Product Quality Attributes through a Multi-Attribute Method (MAM) Lab-of-the-Future. Webinar, Thermo Fisher Scientific; 2020.
• Advances in Biopharmaceutical Characterization eBook Series. Thermo Fisher Scientific; 2020.
• Rogers RS et al. A View on the Importance of ‘Multi-Attribute Method’ for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy. AAPS J. 2018;20(7):1–7.