Abstract
The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry (LC-MS)-based method that is used to directly characterize and monitor numerous product quality attributes (PQAs) at the amino acid level of a biopharmaceutical product. MAM enables identity testing based on primary sequence verification, detection and quantitation of post-translational modifications and impurities. This ability to simultaneously and directly determine PQAs of therapeutic proteins makes MAM a more informative, streamlined and productive workflow than conventional chromatographic and electrophoretic assays. MAM relies on proteolytic digestion of the sample followed by reversed-phase chromatographic separation and high-resolution LC-MS analysis in two phases. First, a discovery study to determine quality attributes for monitoring is followed by the creation of a targeted library based on high-resolution retention time plus accurate mass analysis. The second aspect of MAM is the monitoring phase based on the target peptide library and new peak detection using differential analysis of the data to determine the presence, absence or change of any species that might affect the activity or stability of the biotherapeutic. The sample preparation process takes between 90 and 120 min, whereas the time spent on instrumental and data analyses might vary from one to several days for different sample sizes, depending on the complexity of the molecule, the number of attributes to be monitored and the information to be detailed in the final report. MAM is developed to be used throughout the product life cycle, from process development through upstream and downstream processes to quality control release or under current good manufacturing practices regulations enforced by regulatory agencies.
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Data availability
All LC-MS data used in this paper are publicly available at the GNPS-MassIVE repository under the following accession numbers: MSV000088774 (chimeric IgG1 monoclonal antibody drug product; three replicates are provided as 1, 2 and 3); MSV000088775 (chimeric IgG1 monoclonal antibody drug product spiked with different levels of HCPs, cathepsin and rHLPL, at three different levels: 10, 100 and 1,000 ppm; three replicates are provided for each spiked level as 1, 2 and 3); and MSV000089060 (chimeric IgG1 monoclonal antibody investigational biosimilar; three replicates are provided as 1, 2 and 3).
References
Yasunaga, M. Antibody therapeutics and immunoregulation in cancer and autoimmune disease. Semin. Cancer Biol. 64, 1–12 (2020).
Shim, H. Bispecific antibodies and antibody-drug conjugates for cancer therapy: technological considerations. Biomolecules 10, 360 (2020).
Shu, S. A., Wang, J., Tao, M. H. & Leung, P. S. Gene therapy for autoimmune disease. Clin. Rev. Allergy Immunol. 49, 163–176 (2015).
Islam, M. A., Kundu, S. & Hassan, R. Gene therapy approaches in an autoimmune demyelinating disease: multiple sclerosis. Curr. Gene Ther. 19, 376–385 (2020).
World Preview 2018, Outlook to 2024. Evaluate Pharma, 1–47 Available at https://www.evaluate.com/thought-leadership/pharma/evaluatepharma-world-preview-2018-outlook-2024?gclid=Cj0KCQiAmaibBhCAARIsAKUlaKTZfRwuRaj58cikLdbczZL8vgBdGJpJX2DJ2GQKWpFuGn8BKbN9ZWAaAm8OEALw_wcB (2018).
Mullard, A. FDA approves 100th monoclonal antibody product. Nat. Rev. Drug Discov. 20, 491–495 (2021).
Jefferis, R. Posttranslational modifications and the immunogenicity of biotherapeutics. J. Immunol. Res. 2016, 5358272 (2016).
Xu, Y. et al. Structure, heterogeneity and developability assessment of therapeutic antibodies. MAbs 11, 239–264 (2019).
Virág, D. et al. Current trends in the analysis of post‑translational modifications. Chromatographia 83, 1–10 (2020).
Graf, T. et al. Recent advances in LC-MS based characterization of protein-based bio-therapeutics - mastering analytical challenges posed by the increasing format complexity. J. Pharm. Biomed. Anal. 186, 113251 (2020).
Rogstad, S. et al. A retrospective evaluation of the use of mass spectrometry in FDA biologics license applications. J. Am. Soc. Mass Spectrom. 28, 786–794 (2017).
Apostol, I. et al. Enabling development, manufacturing, and regulatory approval of biotherapeutics through advances in mass spectrometry. Curr. Opin. Biotehcnol. 71, 206–215 (2021).
Rogers, R. S. et al. Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics. MAbs 7, 881–890 (2015).
Rogers, R. S. et al. A view on the importance of “multi-attribute method” for measuring purity of biopharmaceuticals and improving overall control strategy. AAPS J. 20, 7 (2018).
Xu, W. et al. A quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins. MAbs 9, 1186–1196 (2017).
Rogstad, S. et al. Multi-attribute method for quality control of therapeutic proteins. Anal. Chem. 91, 14170–14177 (2019).
Buettner, A., Maier, M., Bonnington, L., Bulau, P. & Reusch, D. Multi-attribute monitoring of complex erythropoetin beta glycosylation by GluC liquid chromatography-mass spectrometry peptide mapping. Anal. Chem. 92, 7574–7580 (2020).
Liu, Y. et al. Simultaneous monitoring and comparison of multiple product quality attributes for cell culture processes at different scales using a LC/MS/MS based multi-attribute method. J. Pharm. Sci. 109, 1–11 (2020).
Ren, D. Advancing mass spectrometry technology in cGMP environments. Trends Biotechnol. 38, 1051–1053 (2020).
Sokolowska, I. et al. Implementation of a high-resolution liquid chromatography-mass spectrometry method in quality control laboratories for release and stability testing of a commercial antibody product. Anal. Chem. 92, 2369–2373 (2020).
Tajiri-Tsukada, M., Hashii, N. & Ishii-Watabe, A. Establishment of a highly precise multi-attribute method for the characterization and quality control of therapeutic monoclonal antibodies. Bioengineered 11, 984–1000 (2020).
Zhang, Z., Chan, P. K., Richardson, J. & Shah, B. An evaluation of instrument types for mass spectrometry-based multi-attribute analysis of biotherapeutics. MAbs 12, 1783062 (2020).
Mouchahoir, T. et al. New peak detection performance metrics from the MAM Consortium Interlaboratory Study. J. Am. Soc. Mass Spectrom. 32, 913–928 (2021).
Song, Y. E. et al. Automated mass spectrometry multi-attribute method analyses for process development and characterization of mAbs. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1166, 122540 (2021).
Evans, A. R., Hebert, A. S., Mulholland, J., Lewis, M. J. & Hu, P. ID-MAM: a validated identity and multi-attribute monitoring method for commercial release and stability testing of a bispecific antibody. Anal. Chem. 93, 9166––9173 (2021).
Jakes, C. et al. Tracking the behavior of monoclonal antibody product quality attributes using a multi-attribute method workflow. J. Am. Soc. Mass Spectrom. 32, 1998–2012 (2021).
Atouf, F., Chess, E. & McCarthy, D. Shaping tomorrow’s solutions to today’s biologics quality challenges. USP Biologics Open Forum, April 28, 2021. Available at https://www.usp.org/get-involved/provide-input/stakeholder-forums/bio-mam (2021).
Liu, Y. et al. A fully integrated online platform for real time monitoring of multiple product quality attributes in biopharmaceutical processes for monoclonal antibody therapeutics. J. Pharm. Sci. 111, 358–367 (2022).
Melmer, M. et al. HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals. Anal. Bioanal. Chem. 398, 905–914 (2010).
Fekete, S., Beck, A., Veuthey, J. L. & Guillarme, D. Ion-exchange chromatography for the characterization of biopharmaceuticals. J. Pharm. Biomed. Anal. 113, 43–55 (2015).
Rustandi, R. R., Washabaugh, M. W. & Wang, Y. Applications of CE SDS gel in development of biopharmaceutical antibody-based products. Electrophoresis 29, 3612–3620 (2008).
Wang, Y. et al. Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion. MAbs 8, 1477–1486 (2016).
Wang, T. et al. Application of a quantitative LC-MS multiattribute method for monitoring site-specific glycan heterogeneity on a monoclonal antibody containing two N-linked glycosylation sites. Anal. Chem. 89, 3562–3567 (2017).
Bomans, K. et al. Multi-attribute monitoring of antibody modifications by semi-automated liquid chromatography mass spectrometry peptide mapping. Am. Pharm. Rev. Available at https://www.americanpharmaceuticalreview.com/Featured-Articles/331529-Multi-Attribute-Monitoring-of-Antibody-Modifications-by-Semi-Automated-Liquid-Chromatography-Mass-Spectrometry-Peptide-Mapping/ (2016).
Ogata, Y. et al. eAutomated multi-attribute method sample preparation using high-throughput buffer exchange tips. Rapid Commun. Mass Spectrom. 36, e9222. (2021).
Hao, Z. et al. Multi-attribute method performance profile for quality control of monoclonal antibody therapeutics. J. Pharm. Biomed. Anal. 205, 114330 (2021).
Dong, J. et al. High-throughput, automated protein A purification platform with multiattribute LC-MS analysis for advanced cell culture process monitoring. Anal. Chem. 88, 8673–8679 (2016).
Millán-Martín, S. et al. Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes. Anal. Bioanal. Chem. 412, 6833–6848 (2020).
Arndt, J. R. et al. High-resolution ion-mobility-enabled peptide mapping for high-throughput critical quality attribute monitoring. J. Am. Soc. Mass Spectrom. 32, 2019–2032 (2021).
Jakes, C. et al. Consistent results for peptide mapping and monitoring across three systems of the Vanquish UHPLC platform (AN-001123). Thermo Fisher Scientific Available at https://www.thermofisher.com/es/en/home/products-and-services/promotions/industrial/simplify-biopharma-hr-mam-workflow.html (2022).
Qian, C., Niu, B., Jimenez, R. B., Wang, J. & Albarghouthi, M. Fully automated peptide mapping multi-attribute method by liquid chromatography-mass spectrometry with robotic liquid handling system. J. Pharm. Biomed. Anal. 198, 113988 (2021).
Rogers, R., Swenson, S., Khuu-Duong, K. & Zhang, T. Mass spectrometry-based process analytical technologies for cell therapies. MAM Consortium Available at http://mamconsortium.org/ (2021).
Carillo, S., Criscuolo, A., Fussl, F., Cook, K. & Bones, J. Intact multi-attribute method (iMAM): a flexible tool for the analysis of monoclonal antibodies. Eur. J. Pharm. Biopharm. 177, 241–248 (2022).
Yu, L. X. et al. Understanding pharmaceutical quality by design. AAPS J. 16, 771–783 (2014).
Zhang, Y. & Yang, H. Bringing Multi-Attribute Method (MAM) to the next level. ASMS 2021, Pennsylvania Convention Center (November 2021). Available at https://www.asms.org/publications/abstracts-and-proceedings (2021).
Yang, H. et al. The MAM 2.0 workflow enables seamless transition from research and development to quality control (AN000463). Thermo Fisher Scientific Available at https://www.thermofisher.com/es/en/home/products-and-services/promotions/industrial/simplify-biopharma-hr-mam-workflow.html (2022).
Robinson, C. J. & Jones, C. Quality control and analytical techniques for biopharmaceuticals. Bioanalysis 3, 81–95 (2011).
Beck, A., Wagner-Rousset, E., Ayoub, D., Van Dorsselaer, A. & Sanglier-Cianferani, S. Characterization of therapeutic antibodies and related products. Anal. Chem. 85, 715–736 (2013).
Berkowitz, S. A., Engen, J. R., Mazzeo, J. M. & Jones, G. B. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat. Rev. Drug Discov. 11, 527–540 (2012).
Zhang, X., Chemmalil, L., Ding, J., Mussa, N. & Li, Z. Imaged capillary isoelectric focusing in native condition: a novel and successful example. Anal. Biochem. 537, 13–19 (2017).
Zhang, L. et al. Characterization and elimination of artificial non-covalent light Chain dimers in reduced CE-SDS analysis of pertuzumab. J. Pharm. Biomed. Anal. 190, 113527 (2020).
Esterman, A. L., Katiyar, A. & Krishnamurthy, G. Implementation of USP antibody standard for system suitability in capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for release and stability methods. J. Pharm. Biomed. Anal. 128, 447–454 (2016).
Fekete, S., Guillarme, D., Sandra, P. & Sandra, K. Chromatographic, electrophoretic, and mass spectrometric methods for the analytical characterization of protein biopharmaceuticals. Anal. Chem. 88, 480–507 (2016).
Zhu-Shimoni, J. et al. Host cell protein testing by ELISAs and the use of orthogonal methods. Biotechnol. Bioeng. 111, 2367–2379 (2014).
EMA. ICH Topic Q 6 B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. Available at https://www.ema.europa.eu/en/documents/scientific-guideline/ich-q-6-b-test-procedures-acceptance-criteria-biotechnological/biological-products-step-5_en.pdf (1999).
Sitasuwan, P. et al. Enhancing the multi-attribute method through an automated and high-throughput sample preparation. MAbs 13, e1978131 (2021).
Dong, M. W. Tryptic mapping by reversed phase liquid chromatography. Adv. Chromatogr. 32, 21–51 (1992).
Cho, I. H. et al. Evaluation of the structural, physicochemical, and biological characteristics of SB4, a biosimilar of etanercept. MAbs 8, 1136–1155 (2016).
Stavenhagen, K. et al. Site-specific N- and O-glycosylation analysis of atacicept. MAbs 11, 1053–1063 (2019).
Guapo, F., Strasser, L., Millán-Martín, S., Anderson, I. & Bones, J. Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs). J. Pharm. Biomed. Anal. 207, 1–7 (2021).
Toole, E. N. et al. Rapid highly-efficient digestion and peptide mapping of adeno-associated viruses. Anal. Chem. 93, 10403–10410 (2021).
Ren, D. et al. An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Anal. Biochem. 392, 12–21 (2009).
Zhou, M., Gucinski, A. C. & Boyne, M. T. 2nd Performance metrics for evaluating system suitability in liquid chromatography—Mass spectrometry peptide mass mapping of protein therapeutics and monoclonal antibodies. MAbs 7, 1104–1117 (2015).
FDA. Part 11, Electronic Records; Electronic Signatures - Scope and Application. Guidance for Industry. (September 2003). Available at https://www.fda.gov/regulatory-information/search-fda-guidance-documents/part-11-electronic-records-electronic-signatures-scope-and-application (2003).
Mesmin, C., Manache-Alberici, L. & Jones, J. Validation of LC-MS Multi-Attribute Method (MAM) Supporting Biopharma Process Characterization. Available at https://www.biopharminternational.com/view/validation-lc-ms-multi-attribute-method-mam-supporting-biopharma-process-characterization (2020).
Rogers, R. Case Study: Automated Software-Driven Implementation of the Multi-Attribute Method (MAM) for Biotherapeutic Characterization. Available at https://www.genedata.com/fileadmin/4.Resources/Case-studies/Ex_Just_MAM_19E03.pdf (2019).
Dong, Q. et al. The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization. MAbs 10, 354–369 (2018).
Schiel, J. E., Davis, D. L. & Borisov, O. V. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The NISTmAb Case Study. ACS Symposium Series. Vol. 1201 Available at https://pubs.acs.org/doi/10.1021/bk-2015-1201 (2015).
Mouchahoir, T., Rogers, R. & Schiel, J. Interlaboratory performance metrics from the MAM Consortium New Peak Detection Round Robin Study. 15th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry (San Francisco, CA) Available at https://www.nist.gov/publications/new-peak-detection-performance-metrics-mam-consortium-interlaboratory-study (2018).
Rogers, R. MS in QC: a single multi-attribute method for quality control and release testing of biologics. CASSS MS, Boston, 24 September 2013.
Yandrofski, K. et al. Interlaboratory studies using the NISTmAb to advance biopharmaceutical structural analytics. Front. Mol. Biosci. 9, 876780 (2022).
Acknowledgements
The authors gratefully acknowledge Thermo Fisher Scientific for instrument access and financial support.
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C.J. and S.C. codeveloped the analytical method, acquired data and reviewed and edited the manuscript. S.M.-M. performed data analysis and interpretation and wrote the manuscript. J.B. codeveloped the analytical method, performed data interpretation, edited the manuscript and was the academic lead. R.R. and D.R. contributed to the writing and edited the manuscript.
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J.B. received funding to support undertaking this study as part of a funded collaboration between NIBRT and Thermo Fisher Scientific. S.C., C.J. and S.M.-M. are employed on this collaborative project. Beyond this, the authors are not aware of any affiliations, memberships, funding or financial holdings that might be perceived as affecting the objectivity of this article. The authors declare no competing financial interests.
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Jakes, C. J. Am. Soc. Mass Spectrom. 32, 1998–2012 (2021): https://doi.org/10.1021/jasms.0c00432
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Supplementary Figs. 1–4 and Table 1
Supplementary Data 1
Data processing walk-through details
Supplementary Data 2
SST report obtained for Hypersil Gold 150 × 2.1-mm, 3-µm column, including all evaluated parameters and monitored quality attributes
Supplementary Data 3
SST report obtained for Acclaim C18, 250 × 2.1-mm, 2.2-µm column, including all evaluated parameters and monitored quality attributes
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Millán-Martín, S., Jakes, C., Carillo, S. et al. Comprehensive multi-attribute method workflow for biotherapeutic characterization and current good manufacturing practices testing. Nat Protoc 18, 1056–1089 (2023). https://doi.org/10.1038/s41596-022-00785-5
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DOI: https://doi.org/10.1038/s41596-022-00785-5
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