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Antibodies
Volume 13
Issue 2
10.3390/antib13020030
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Article

Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation

by
Eleanor Watts
1,
Aarti Bashyal
1,
Sean D. Dunham
1,
Christopher M. Crittenden
2 and
Jennifer S. Brodbelt
1,*
1
Department of Chemistry, University of Texas at Austin, Austin, TX 78712, USA
2
Small Molecule Pharmaceutical Sciences, Genentech Inc., South San Francisco, CA 94080, USA
*
Author to whom correspondence should be addressed.
Antibodies 2024, 13(2), 30; https://doi.org/10.3390/antib13020030
Submission received: 20 February 2024 / Revised: 2 April 2024 / Accepted: 11 April 2024 / Published: 17 April 2024
(This article belongs to the Section Antibody-Based Therapeutics)
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Abstract

As the development of new biotherapeutics advances, increasingly sophisticated tandem mass spectrometry methods are needed to characterize the most complex molecules, including antibody drug conjugates (ADCs). Lysine-linked ADCs, such as trastuzumab-emtansine (T-DM1), are among the most heterogeneous biotherapeutics. Here, we implement a workflow that combines limited proteolysis with HCD-triggered EThcD and UVPD mass spectrometry for the characterization of the resulting middle-down large-sized peptides of T-DM1. Fifty-three payload-containing peptides were identified, ranging in mass from 1.8 to 16.9 kDa, and leading to the unambiguous identification of 46 out of 92 possible conjugation sites. In addition, seven peptides were identified containing multiple payloads. The characterization of these types of heterogeneous peptides represents an important step in unraveling the combinatorial nature of lysine-conjugated ADCs.
Keywords: antibody drug conjugate; trastuzumab-emtansine; tandem mass spectrometry; payload; biotherapeutic antibody drug conjugate; trastuzumab-emtansine; tandem mass spectrometry; payload; biotherapeutic
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MDPI and ACS Style

Watts, E.; Bashyal, A.; Dunham, S.D.; Crittenden, C.M.; Brodbelt, J.S. Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation. Antibodies 2024, 13, 30. https://doi.org/10.3390/antib13020030

AMA Style

Watts E, Bashyal A, Dunham SD, Crittenden CM, Brodbelt JS. Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation. Antibodies. 2024; 13(2):30. https://doi.org/10.3390/antib13020030

Chicago/Turabian Style

Watts, Eleanor, Aarti Bashyal, Sean D. Dunham, Christopher M. Crittenden, and Jennifer S. Brodbelt. 2024. "Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation" Antibodies 13, no. 2: 30. https://doi.org/10.3390/antib13020030

APA Style

Watts, E., Bashyal, A., Dunham, S. D., Crittenden, C. M., & Brodbelt, J. S. (2024). Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation. Antibodies, 13(2), 30. https://doi.org/10.3390/antib13020030

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