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Article
Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry Yuetian Yan, Anita Liu, Shunhai Wang, Thomas J. Daly, and Ning Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03773 • Publication Date (Web): 03 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018
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Analytical Chemistry
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Ultrasensitive
Characterization
of
Charge
Heterogeneity
of
Therapeutic
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Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled
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to Native Mass Spectrometry
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Yuetian Yan, Anita P. Liu, Shunhai Wang, Thomas J. Daly and Ning Li
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Analytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, U.S.A
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CONTACT Shunhai Wang
[email protected] Regeneron Pharmaceuticals Inc.,
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777 Old Saw Mill River Road, Tarrytown, NY, 10591-6707
8 Disclosures
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Yuetian Yan, Anita P. Liu, Shunhai Wang, Thomas J. Daly and Ning Li are full-time employees
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and shareholders of Regeneron Pharmaceuticals Inc.
13 Acknowledgement
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This study was sponsored by Regeneron Pharmaceuticals Inc. The authors would like to thank
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Katherine Pochini and Ashley Roberts of Scientific Writing Group for their assistance in drafting
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and editing the manuscript.
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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Abstract
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In therapeutic monoclonal antibody (mAb) development, charge heterogeneity of a mAb
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molecule is often associated with a critical quality attribute and is therefore monitored
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throughout development and during QC release to ensure product and process consistency.
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Elucidating the cause of each charge variant species is an involved process that often requires
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offline fractionation by ion exchange chromatography (IEX) followed by mass spectrometry (MS)
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analysis, largely due to the incompatibility of conventional IEX buffers for direct MS detection. In
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this study, we have developed a method that combines a generic strong cation exchange
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chromatography (SCX) step with ultrasensitive online native MS analysis (SCX-MS) optimized
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for mAb separation and detection. As demonstrated by analyzing mAb molecules with a wide
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range of pI (isoelectric point) values, the developed method can consistently achieve both high-
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resolution IEX separation and ultrasensitive MS detection of low-abundance charge variant
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species. Using this method, we analyzed the charge heterogeneity of NISTmAb reference
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material 8671 (NISTmAb) at both whole antibody and subdomain levels. In particular, due to the
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high sensitivity, a non-consensus Fab glycosylation site, present at a very low level (
